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Mulberrin ameliorates hepatic dysfunction in CCl 4 -induced mice. (A) The experiment design scheme. (B) Body weights of each group of mice. (C) Survival rate of mice from all groups was quantified. Serum (D) TBIL, (E) ALT, AST, ALP, and (F) LDH levels were examined. Representative data were expressed as mean ± SEM (n = 10 per group). **P < 0.01 and ***P < 0.001 vs the Oil/Veh group; +P < 0.05 and ++P < 0.01 vs the CCl 4 /Veh group.
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Mulberrin (Mul) is a key component of the traditional Chinese medicine Romulus Mori with various biological functions. However, the effects of Mul on liver fibrosis have not been addressed, and thus were investigated in our present study, as well as the underlying mechanisms. Here, we found that Mul administration significantly ameliorated carbon t...
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... TRIM31 deletion (TRIM31 Hep-cKO ) mice were produced by mating TRIM31 Flox/Flox mice with albumin-Cre (Alb-Cre) mice (Jackson Laboratory, Bar Harbor, Maine, USA). A simple schematic diagram has been indicated in Supplementary Fig. S13A. TRIM31 Flox/Flox (Flox) mice littermates were used in the work as controls for the obtained TRIM31 Hep-cKO mice. ...
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... #S-119; HPLC>98%; Chengdu Herbpurify CO., LTD, Chengdu, China) at 15, 30 or 60 mg/kg was administered to each group of mice by gavage daily. The dosages of Mul for animal treatment were referred to previous studies and our preliminary experiments [25,26]. Vehicle groups of mice received 0.9% saline. Animal experimental protocols were presented in Fig. 1A. During the treatments, body weights of mice were recorded weekly. Survival rates of mice were measured. Blood samples were taken either from the tail vein or via the cardiac puncture. The sampled blood was collected into EDTA-coated tubes through retro-orbital bleeding, and centrifuged at 5000 rpm for 10 min at 4 • C. The supernatant ...
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... the present study, a mouse model with hepatic fibrosis was established using CCl 4 to explore the regulatory effects of Mul on liver injury (Fig. 1A). As displayed in Fig. 1B, CCl 4 injection significantly reduced the body weights of mice, but were moderately rescued by Mul at higher dosages (30 and 60 mg/kg). CCl 4 treatment led to poorer survival rates of mice over the course of the experiment compared with Oil/Veh group, while Mul treatments reduced the mortality of CCl 4 ...
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... the present study, a mouse model with hepatic fibrosis was established using CCl 4 to explore the regulatory effects of Mul on liver injury (Fig. 1A). As displayed in Fig. 1B, CCl 4 injection significantly reduced the body weights of mice, but were moderately rescued by Mul at higher dosages (30 and 60 mg/kg). CCl 4 treatment led to poorer survival rates of mice over the course of the experiment compared with Oil/Veh group, while Mul treatments reduced the mortality of CCl 4 -challenged mice (Fig. 1C). CCl ...
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... As displayed in Fig. 1B, CCl 4 injection significantly reduced the body weights of mice, but were moderately rescued by Mul at higher dosages (30 and 60 mg/kg). CCl 4 treatment led to poorer survival rates of mice over the course of the experiment compared with Oil/Veh group, while Mul treatments reduced the mortality of CCl 4 -challenged mice (Fig. 1C). CCl 4 injection significantly induced the hepatic dysfunction and injury in mice, as evidenced by the increased serum TBIL, ALT, AST, ALP and LDH contents; however, all these results caused by CCl 4 were efficiently reversed by Mul treatments in a dosedependent manner (Fig. 1D-F). These findings initially revealed the protective ...
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... while Mul treatments reduced the mortality of CCl 4 -challenged mice (Fig. 1C). CCl 4 injection significantly induced the hepatic dysfunction and injury in mice, as evidenced by the increased serum TBIL, ALT, AST, ALP and LDH contents; however, all these results caused by CCl 4 were efficiently reversed by Mul treatments in a dosedependent manner (Fig. 1D-F). These findings initially revealed the protective effects of Mul against CCl 4 -triggered hepatic damage. Furthermore, we showed that under normal physiological conditions, Mul at 60 mg/kg had no significant influences on the changes of body weights, animal survival and hepatic functions, indicating the safe use of Mul in ...
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... in vivo results indicated the protective effects of Mul against fibrosis, inflammation and oxidative stress in CCl 4 -treated mice. To confirm the function of Mul, in vitro experiments were subsequently performed using human or mouse hepatocyte and HSC cell lines. At first, CCK-8 analysis was used to examine the safe use of Mul. As displayed in Supplementary Figs. 1A and B, Mul treatments at various concentrations had no significant influences on the survival of human HSC cell line LX-2 and hepatocyte L02, respectively, indicating the non-cytotoxicity of Mul. Considering that TGF-β1 plays an important role in hepatic fibrosis and is an inducer of the fibrotic response [36], TGF-β1 was then exposed ...
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... which were significantly and dose-dependently decreased by Mul, particularly starting from 10 μM (Fig. 7A). Thereafter, 25 μM (low dosage), 50 μM (moderate concentration) and 100 μM (high dosage) of Mul were used for further in vitro studies. CCK-8 results confirmed the safe use of 100 μM Mul treatment from 0 to 72 h both in LX-2 and L02 cells ( Supplementary Figs. 1C and D). IF analysis then validated that Mul treatments reduced α-SMA expression in LX-2 cells after TGF-β1 stimulation ( Fig. 7B and ...
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... chosen for subsequent analysis. DCF-DA staining indicated that in TGF-β1-stimulated L02 cells, Mul-ameliorated ROS generation was significantly diminished upon Nrf2 silence (Supplementary Fig. 10A). Consistently, H 2 O 2 and MDA levels restrained by Mul were also restrengthened by siNrf2 in TGF-β1-exposed L02 cells ( Supplementary Figs. ...
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... chosen for subsequent analysis. DCF-DA staining indicated that in TGF-β1-stimulated L02 cells, Mul-ameliorated ROS generation was significantly diminished upon Nrf2 silence (Supplementary Fig. 10A). Consistently, H 2 O 2 and MDA levels restrained by Mul were also restrengthened by siNrf2 in TGF-β1-exposed L02 cells ( Supplementary Figs. 10B and C). In contrast, siNrf2 markedly eliminated the capacity of Mul to improve SOD and GSH levels in L02 cells under TGF-β1 stimuli ( Supplementary Figs. 10D and E), along with decreased HO-1 and NQO1 expression levels (Supplementary Fig. 10F). In hepatocytes with TGF-β1 exposure, Mul-suppressed NOX2 and NOX4 gene expression levels were ...
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... significantly diminished upon Nrf2 silence (Supplementary Fig. 10A). Consistently, H 2 O 2 and MDA levels restrained by Mul were also restrengthened by siNrf2 in TGF-β1-exposed L02 cells ( Supplementary Figs. 10B and C). In contrast, siNrf2 markedly eliminated the capacity of Mul to improve SOD and GSH levels in L02 cells under TGF-β1 stimuli ( Supplementary Figs. 10D and E), along with decreased HO-1 and NQO1 expression levels (Supplementary Fig. 10F). In hepatocytes with TGF-β1 exposure, Mul-suppressed NOX2 and NOX4 gene expression levels were almost abolished upon Nrf2 deletion (Supplementary Fig. 10G). Western blotting confirmed that after TGF-β1 stimulation, Nrf2 was almost undetectable both in ...
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... contrast, siNrf2 markedly eliminated the capacity of Mul to improve SOD and GSH levels in L02 cells under TGF-β1 stimuli ( Supplementary Figs. 10D and E), along with decreased HO-1 and NQO1 expression levels (Supplementary Fig. 10F). In hepatocytes with TGF-β1 exposure, Mul-suppressed NOX2 and NOX4 gene expression levels were almost abolished upon Nrf2 deletion (Supplementary Fig. 10G). ...
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... and E), along with decreased HO-1 and NQO1 expression levels (Supplementary Fig. 10F). In hepatocytes with TGF-β1 exposure, Mul-suppressed NOX2 and NOX4 gene expression levels were almost abolished upon Nrf2 deletion (Supplementary Fig. 10G). Western blotting confirmed that after TGF-β1 stimulation, Nrf2 was almost undetectable both in nucleus and cytoplasm either with or without Mul exposure (Supplementary Fig. 10H). ...
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... hepatocytes with TGF-β1 exposure, Mul-suppressed NOX2 and NOX4 gene expression levels were almost abolished upon Nrf2 deletion (Supplementary Fig. 10G). Western blotting confirmed that after TGF-β1 stimulation, Nrf2 was almost undetectable both in nucleus and cytoplasm either with or without Mul exposure (Supplementary Fig. 10H). Taken together, all these data depicted that Mul exerted antioxidant function mainly through improving Nrf2 signaling. ...
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... that the inflammatory response and oxidative stress in hepatocytes are crucial for HSCs activation and fibrosis, conditional medium (CM) derived from L02 cells was collected and subjected to LX-2 culture to further explore the underlying mechanisms (Fig. 10A). CM from TGF-β1-treated L02 cells markedly increased the expression of α-SMA, Col1a1 and Col3a1 in LX-2 cells, which was significantly ameliorated by Mul (Fig. 10B). IF staining by α-SMA indicated that CM derived from TGF-β1-exposed L02 cells led to HSCs activation, while being greatly ameliorated upon Mul treatment (Fig. 10C and D). ...
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... are crucial for HSCs activation and fibrosis, conditional medium (CM) derived from L02 cells was collected and subjected to LX-2 culture to further explore the underlying mechanisms (Fig. 10A). CM from TGF-β1-treated L02 cells markedly increased the expression of α-SMA, Col1a1 and Col3a1 in LX-2 cells, which was significantly ameliorated by Mul (Fig. 10B). IF staining by α-SMA indicated that CM derived from TGF-β1-exposed L02 cells led to HSCs activation, while being greatly ameliorated upon Mul treatment (Fig. 10C and D). CM obtained from TGF-β1-stimulated L02 cells significantly promoted the TGF-β1/SMADs signaling in LX-2 cells, which was almost diminished after Mul treatment, as ...
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... mechanisms (Fig. 10A). CM from TGF-β1-treated L02 cells markedly increased the expression of α-SMA, Col1a1 and Col3a1 in LX-2 cells, which was significantly ameliorated by Mul (Fig. 10B). IF staining by α-SMA indicated that CM derived from TGF-β1-exposed L02 cells led to HSCs activation, while being greatly ameliorated upon Mul treatment (Fig. 10C and D). CM obtained from TGF-β1-stimulated L02 cells significantly promoted the TGF-β1/SMADs signaling in LX-2 cells, which was almost diminished after Mul treatment, as indicated by the decreased expression of TGF-β1, p-SMAD2/3 and α-SMA (Fig. 10E). These findings elucidated that under fibrotic stresses, hepatocyte damage contributed to HSCs ...
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... TGF-β1-exposed L02 cells led to HSCs activation, while being greatly ameliorated upon Mul treatment (Fig. 10C and D). CM obtained from TGF-β1-stimulated L02 cells significantly promoted the TGF-β1/SMADs signaling in LX-2 cells, which was almost diminished after Mul treatment, as indicated by the decreased expression of TGF-β1, p-SMAD2/3 and α-SMA (Fig. 10E). These findings elucidated that under fibrotic stresses, hepatocyte damage contributed to HSCs activation, and such effect could be abolished by ...
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... levels in LX-2 cells, which were, however, accelerated upon TRIM31 and Nrf2 deletion. Additionally, in LX-2 cells exposed to CM from TGF-β1-stimulated L02 cells, Mul-inhibited expression of these fibrotic genes was considerably abolished by siTRIM31 and siNrf2 ( (caption on next page) expression of TGF-β1, p-SMAD2/3 and α-SMA in LX-2 cells (Fig. 11D and E). Collectively, these findings demonstrated that the TRIM31-Nrf2 axis in hepatocytes was crucial for Mul to restrain HSCs activation in ...
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... CM derived from TGF-β1-incubated L02 cells led to higher contents of IL-1β, TNF-α, IL-18 and CXCL-10 in supernatants, which were significantly accelerated upon TRIM31 and Nrf2 knockdown. Similarly, Mul-reduced concentrations of these inflammatory factors were markedly restrengthened by siTRIM31 or siNrf2 (Fig. ...
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... deletion. Mul-improved expression of TRIM31 and Nrf2 was also significantly abolished when TRIM31 and Nrf2 were ablated. In contrast, NLRP3, ASC, Caspase-1 and p-NF-κB protein expression levels potentiated by CM-TGF-β1 were further exacerbated by siTRIM31 and siNrf2. Consistently, Mul-inhibited expression of these signals was strongly restored Fig. 11. Mulberrin inhibits fibrosis via the improvements of TRIM31 and Nrf2 signaling pathways. L02 cells were transfected with siTRIM31-1# or siNRf2-2# for 24 h for the knockdown of TRIM31 and Nrf2, respectively, and were then stimulated by TGF-β1 (10 ng/ml) for another 24 h in the absence or presence of Mul-H (100 μM). The cultured medium ...
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... mean ± SEM (n = 3 per group). Scale bar, 10 μm. **P < 0.01 and ***P < 0.001 vs the CM-Ctrl group; + P < 0.05 and ++ P < 0.01 vs the CM-TGF-β1 group; # P < 0.05 and ## P < 0.01. Representative data were expressed as mean ± SEM (n = 9 per group). # P < 0.05, ## P < 0.01 and ### P < 0.001; ns, no significant difference. upon TRIM31 and Nrf2 silence (Fig. 11G). The expression changes of TRIM31/NLRP3 and Nrf2 signaling in LX-2 cells might be attributed to the severer inflammatory ...
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... confirm the interaction between hepatocytes and HSCs mediated by Mul under fibrotic stresses in vivo, the hepatocyte-specific TRIM31 knockout (TRIM31 Hep-cKO ) mice were generated ( Supplementary Fig. 11A). Western blotting results confirmed that TRIM31 was not detected in liver of TRIM31 Hep-cKO mice ( Supplementary Figs. ...
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... confirm the interaction between hepatocytes and HSCs mediated by Mul under fibrotic stresses in vivo, the hepatocyte-specific TRIM31 knockout (TRIM31 Hep-cKO ) mice were generated ( Supplementary Fig. 11A). Western blotting results confirmed that TRIM31 was not detected in liver of TRIM31 Hep-cKO mice ( Supplementary Figs. 11B and C). H&E staining demonstrated that there were no evident histological changes in liver sections between TRIM31 Flox and TRIM31 Hep-cKO groups of mice (Supplementary Fig. 11D). IF staining validated the successful TRIM31 deletion in liver of TRIM31 Hep-cKO mice ( Supplementary Fig. 11E). In response to CCl 4 , Mul improved the body weights ...
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... and C). H&E staining demonstrated that there were no evident histological changes in liver sections between TRIM31 Flox and TRIM31 Hep-cKO groups of mice (Supplementary Fig. 11D). IF staining validated the successful TRIM31 deletion in liver of TRIM31 Hep-cKO mice ( Supplementary Fig. 11E). ...
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... staining demonstrated that there were no evident histological changes in liver sections between TRIM31 Flox and TRIM31 Hep-cKO groups of mice (Supplementary Fig. 11D). IF staining validated the successful TRIM31 deletion in liver of TRIM31 Hep-cKO mice ( Supplementary Fig. 11E). In response to CCl 4 , Mul improved the body weights of mice, which was, however, abolished in TRIM31 Hep-cKO mice (Fig. 12A). ...
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... histological changes in liver sections between TRIM31 Flox and TRIM31 Hep-cKO groups of mice (Supplementary Fig. 11D). IF staining validated the successful TRIM31 deletion in liver of TRIM31 Hep-cKO mice ( Supplementary Fig. 11E). In response to CCl 4 , Mul improved the body weights of mice, which was, however, abolished in TRIM31 Hep-cKO mice (Fig. 12A). As shown in Fig. 12B-D, Mul failed to mitigate the elevation of serum TBIL, LDH, ALT, AST and ALP in the CCl 4 /TRIM31 Hep-cKO mice. Likewise, the effects of Mul to reduce fibrotic parameters including hyaluronic acid, PC III and Collagen IV levels were impeded in TRIM31 Hep-cKO mice after Representative data were expressed as mean ± ...
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... in liver sections between TRIM31 Flox and TRIM31 Hep-cKO groups of mice (Supplementary Fig. 11D). IF staining validated the successful TRIM31 deletion in liver of TRIM31 Hep-cKO mice ( Supplementary Fig. 11E). In response to CCl 4 , Mul improved the body weights of mice, which was, however, abolished in TRIM31 Hep-cKO mice (Fig. 12A). As shown in Fig. 12B-D, Mul failed to mitigate the elevation of serum TBIL, LDH, ALT, AST and ALP in the CCl 4 /TRIM31 Hep-cKO mice. Likewise, the effects of Mul to reduce fibrotic parameters including hyaluronic acid, PC III and Collagen IV levels were impeded in TRIM31 Hep-cKO mice after Representative data were expressed as mean ± SEM (n = 6 or 9 per ...
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... in TRIM31 Hep-cKO mice after Representative data were expressed as mean ± SEM (n = 6 or 9 per group). Magnification: × 100. # P < 0.05, ## P < 0.01 and ### P < 0.001; ns, no significant difference. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) CCl 4 challenge (Fig. 12E-G). Furthermore, histological staining suggested that Mul treatment failed to restrain the collagen deposition in liver of TRIM31 Hep-cKO mice induced by CCl 4 , along with restored fibrosis scores (Fig. 13A-D). IF staining confirmed that after CCl 4 injection, the function of Mul to reduce α-SMA and Col-I accumulation in liver sections ...
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... interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) CCl 4 challenge (Fig. 12E-G). Furthermore, histological staining suggested that Mul treatment failed to restrain the collagen deposition in liver of TRIM31 Hep-cKO mice induced by CCl 4 , along with restored fibrosis scores (Fig. 13A-D). IF staining confirmed that after CCl 4 injection, the function of Mul to reduce α-SMA and Col-I accumulation in liver sections was almost diminished upon hepatocyte-specific TRIM31 knockout ( Fig. 14A-D). Consistently, Mul failed to repress α-SMA, Col1a1, Col3a1 and TGF-β1 gene expression levels in liver of CCl 4 - tissues of all ...
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... treatment failed to restrain the collagen deposition in liver of TRIM31 Hep-cKO mice induced by CCl 4 , along with restored fibrosis scores (Fig. 13A-D). IF staining confirmed that after CCl 4 injection, the function of Mul to reduce α-SMA and Col-I accumulation in liver sections was almost diminished upon hepatocyte-specific TRIM31 knockout ( Fig. 14A-D). Consistently, Mul failed to repress α-SMA, Col1a1, Col3a1 and TGF-β1 gene expression levels in liver of CCl 4 - tissues of all groups of mice. Representative data were expressed as mean ± SEM (n = 4 per group). Magnification: × 200. # P < 0.05, ## P < 0.01 and ### P < 0.001; ns, no significant difference. treated TRIM31 Hep-cKO mice ...
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... Fig. 14A-D). Consistently, Mul failed to repress α-SMA, Col1a1, Col3a1 and TGF-β1 gene expression levels in liver of CCl 4 - tissues of all groups of mice. Representative data were expressed as mean ± SEM (n = 4 per group). Magnification: × 200. # P < 0.05, ## P < 0.01 and ### P < 0.001; ns, no significant difference. treated TRIM31 Hep-cKO mice (Fig. 14E). Similarly, the effects of Mul to decrease TGF-β1, p-SMAD2/3 and α-SMA protein expression levels were also completely diminished in CCl 4 /TRIM31 Hep-cKO mice (Fig. 14F). Together, these findings elucidated that Mul might exert its anti-fibrotic effects through improvement of hepatocyte TRIM31 ...
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... data were expressed as mean ± SEM (n = 4 per group). Magnification: × 200. # P < 0.05, ## P < 0.01 and ### P < 0.001; ns, no significant difference. treated TRIM31 Hep-cKO mice (Fig. 14E). Similarly, the effects of Mul to decrease TGF-β1, p-SMAD2/3 and α-SMA protein expression levels were also completely diminished in CCl 4 /TRIM31 Hep-cKO mice (Fig. 14F). Together, these findings elucidated that Mul might exert its anti-fibrotic effects through improvement of hepatocyte TRIM31 ...
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... this regard, IHC staining suggested that in response to CCl 4 stimuli, Mul failed to mitigate F4/80 and CD68 positive expression in liver sections of TRIM31 Hep-cKO mice (Fig. 15A and B). TRIM31 Hep-cKO also abolished the capacity of Mul to reduce serum IL-1β, TNF-α and IL-6 contents in CCl 4 -challenged mice (Fig. 15C). Consistently, after CCl 4 Fig. 15. Anti-inflammatory actions of mulberrin in liver fibrosis mice regulated by TRIM31 in vivo. (A) IHC staining for F4/80 and CD68 expression in hepatic sections from ...
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... this regard, IHC staining suggested that in response to CCl 4 stimuli, Mul failed to mitigate F4/80 and CD68 positive expression in liver sections of TRIM31 Hep-cKO mice (Fig. 15A and B). TRIM31 Hep-cKO also abolished the capacity of Mul to reduce serum IL-1β, TNF-α and IL-6 contents in CCl 4 -challenged mice (Fig. 15C). Consistently, after CCl 4 Fig. 15. Anti-inflammatory actions of mulberrin in liver fibrosis mice regulated by TRIM31 in vivo. (A) IHC staining for F4/80 and CD68 expression in hepatic sections from TRIM31 Flox or TRIM31 Hep-cKO mice. (B) F4/80-and CD68-positive areas were quantified after IHC assays. (C) ELISA analysis for serum ...
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... this regard, IHC staining suggested that in response to CCl 4 stimuli, Mul failed to mitigate F4/80 and CD68 positive expression in liver sections of TRIM31 Hep-cKO mice (Fig. 15A and B). TRIM31 Hep-cKO also abolished the capacity of Mul to reduce serum IL-1β, TNF-α and IL-6 contents in CCl 4 -challenged mice (Fig. 15C). Consistently, after CCl 4 Fig. 15. Anti-inflammatory actions of mulberrin in liver fibrosis mice regulated by TRIM31 in vivo. (A) IHC staining for F4/80 and CD68 expression in hepatic sections from TRIM31 Flox or TRIM31 Hep-cKO mice. (B) F4/80-and CD68-positive areas were quantified after IHC assays. (C) ELISA analysis for serum contents of IL-1β, TNF-α and IL-6 from the shown groups of mice. (E) RT-qPCR analysis for IL-1β, TNF-α, IL-6, IL-18, MCP-1 and ...
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... IHC, RT-qPCR and western blotting assays, or 9 for biological analysis per group). Magnification: × 200. # P < 0.05, ## P < 0.01 and ### P < 0.001; ns, no significant difference. injection, hepatic IL-1β, TNF-α, IL-6, IL-18, MCP-1 and CXCL-10 gene expression levels were also failed to be diminished by Mul upon hepatocyte-specific TRIM31 deletion (Fig. 15D). As expected, Mul lost its inhibitory effects on NLRP3, ASC, Caspase-1, p-IKKα, p-IκBα, p-NF-κB, mIL-1β and mIL-18 protein expression levels in liver of CCl 4 / TRIM31 Hep-cKO mice (Fig. 15E). These findings suggested that hepatocyte TRIM31 expression was required for Mul to perform its antiinflammatory ...
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... IL-1β, TNF-α, IL-6, IL-18, MCP-1 and CXCL-10 gene expression levels were also failed to be diminished by Mul upon hepatocyte-specific TRIM31 deletion (Fig. 15D). As expected, Mul lost its inhibitory effects on NLRP3, ASC, Caspase-1, p-IKKα, p-IκBα, p-NF-κB, mIL-1β and mIL-18 protein expression levels in liver of CCl 4 / TRIM31 Hep-cKO mice (Fig. 15E). These findings suggested that hepatocyte TRIM31 expression was required for Mul to perform its antiinflammatory ...
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... further explore whether Nrf2 in hepatocytes was necessary for Mul to control HSCs activation, primary hepatocytes were isolated from the wild type (WT) and Nrf2-knockout (KO) mice and were employed in vitro. Western blotting and IF staining assays confirmed the successful knockout of Nrf2 in the extracted primary mouse hepatocytes (Supplementary Figs. 12A and B). We then established an in vitro CM system derived from Nrf2 WT or Nrf2 KO primary hepatocytes with or without Fig. 16. Anti-fibrotic effect of mulberrin is Nrf2-dependent in the primary HSCs. Primary hepatocytes isolated from Nrf2 WT or Nrf2 KO mice were stimulated by TGF-β1 (10 ng/ml) for 24 h in the absence or presence of Mul-H (100 ...
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... from the wild type (WT) and Nrf2-knockout (KO) mice and were employed in vitro. Western blotting and IF staining assays confirmed the successful knockout of Nrf2 in the extracted primary mouse hepatocytes (Supplementary Figs. 12A and B). We then established an in vitro CM system derived from Nrf2 WT or Nrf2 KO primary hepatocytes with or without Fig. 16. Anti-fibrotic effect of mulberrin is Nrf2-dependent in the primary HSCs. Primary hepatocytes isolated from Nrf2 WT or Nrf2 KO mice were stimulated by TGF-β1 (10 ng/ml) for 24 h in the absence or presence of Mul-H (100 μM). The cultured medium was then collected, and mixed with fresh medium at 1:1 ratio, served as CM. The CM was then ...
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... TGF-β1, p-SMAD2/3 and α-SMA protein expression levels in primary HSCs. Representative data were expressed as mean ± SEM (n = 4 per group). Scale bar, 50 μm. # P < 0.05, ## P < 0.01 and ### P < 0.001; ns, no significant difference. TGF-β1 stimuli, which was then collected for mouse primary HSCs culture in the presence or absence of Mul treatment (Fig. 16A). Firstly, we showed that Mul remarkably improved nucleus Nrf2 expression, but decreased cytoplastic Nrf2 in primary Nrf2 WT hepatocytes after TGF-β1 stimulation. As expected, Nrf2 was undetectable in Nrf2 KO hepatocytes (Fig. 16B). We then found that Mul failed to reduce α-SMA, Col1a1, Col3a1 and Fibronectin expression levels in ...
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... TGF-β1 stimuli, which was then collected for mouse primary HSCs culture in the presence or absence of Mul treatment (Fig. 16A). Firstly, we showed that Mul remarkably improved nucleus Nrf2 expression, but decreased cytoplastic Nrf2 in primary Nrf2 WT hepatocytes after TGF-β1 stimulation. As expected, Nrf2 was undetectable in Nrf2 KO hepatocytes (Fig. 16B). We then found that Mul failed to reduce α-SMA, Col1a1, Col3a1 and Fibronectin expression levels in primary HSCs cultured in CM derived from TGF-β1-incubated Nrf2 KO hepatocytes (Fig. 16C-E). Consistently, the effects of Mul to down-regulate TGF-β1, p-SMAD2/3 and α-SMA protein expression levels were also significantly diminished Fig. ...
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... expression, but decreased cytoplastic Nrf2 in primary Nrf2 WT hepatocytes after TGF-β1 stimulation. As expected, Nrf2 was undetectable in Nrf2 KO hepatocytes (Fig. 16B). We then found that Mul failed to reduce α-SMA, Col1a1, Col3a1 and Fibronectin expression levels in primary HSCs cultured in CM derived from TGF-β1-incubated Nrf2 KO hepatocytes (Fig. 16C-E). Consistently, the effects of Mul to down-regulate TGF-β1, p-SMAD2/3 and α-SMA protein expression levels were also significantly diminished Fig. 17. Exploration of the interaction between TRIM31 and Nrf2. (A,B) The interaction of endogenous TRIM31 with Nrf2 in L02 cells was examined by immunoprecipitation assay using the indicated ...
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... (Fig. 16B). We then found that Mul failed to reduce α-SMA, Col1a1, Col3a1 and Fibronectin expression levels in primary HSCs cultured in CM derived from TGF-β1-incubated Nrf2 KO hepatocytes (Fig. 16C-E). Consistently, the effects of Mul to down-regulate TGF-β1, p-SMAD2/3 and α-SMA protein expression levels were also significantly diminished Fig. 17. Exploration of the interaction between TRIM31 and Nrf2. (A,B) The interaction of endogenous TRIM31 with Nrf2 in L02 cells was examined by immunoprecipitation assay using the indicated antibodies. (C) After TGF-β1 (10 ng/ml) incubation for 24 h, interaction between TRIM31 and Nrf2 in L02 cells was assessed by immunoprecipitation ...
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... and cytoplastic Nrf2 protein expression levels in liver of the indicated groups of mice. Representative data were expressed as mean ± SEM (n = 4 per group). Magnification: × 200. # P < 0.05, ## P < 0.01 and ### P < 0.001; ns, no significant difference. in mouse primary HSCs cultured in CM obtained from Nrf2 KO hepatocytes under TGF-β1 stimuli (Fig. 16F). These data elucidated that the anti-fibrotic effects of Mul were at least in part attributed to hepatocyte Nrf2 ...
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... to further explore the underlying molecular mechanisms, we examined whether there might be a possible interaction between TRIM31 and Nrf2. Endogenous immunoprecipitation and western blotting assays confirmed the interaction between TRIM31 and Nrf2 in human hepatocytes (Fig. 17A and B). Additionally, the interaction between TRIM31 and Nrf2 in L02 cells was weakened upon TGF-β1 stimulation (Fig. 17C). The interaction between TRIM31 and Nrf2 was verified in human HSCs (Fig. 17D and E), and was receded under TGF-β1 stress (Fig. 17F). Moreover, knockdown of TRIM31 expression by siRNA further reduced Nrf2 nuclear ...
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... mechanisms, we examined whether there might be a possible interaction between TRIM31 and Nrf2. Endogenous immunoprecipitation and western blotting assays confirmed the interaction between TRIM31 and Nrf2 in human hepatocytes (Fig. 17A and B). Additionally, the interaction between TRIM31 and Nrf2 in L02 cells was weakened upon TGF-β1 stimulation (Fig. 17C). The interaction between TRIM31 and Nrf2 was verified in human HSCs (Fig. 17D and E), and was receded under TGF-β1 stress (Fig. 17F). Moreover, knockdown of TRIM31 expression by siRNA further reduced Nrf2 nuclear translocation after TGF-β1 treatment in L02 and LX2 cells (Fig. 17G). On the contrary, Nrf2 nuclear expression was ...
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... TRIM31 and Nrf2. Endogenous immunoprecipitation and western blotting assays confirmed the interaction between TRIM31 and Nrf2 in human hepatocytes (Fig. 17A and B). Additionally, the interaction between TRIM31 and Nrf2 in L02 cells was weakened upon TGF-β1 stimulation (Fig. 17C). The interaction between TRIM31 and Nrf2 was verified in human HSCs (Fig. 17D and E), and was receded under TGF-β1 stress (Fig. 17F). Moreover, knockdown of TRIM31 expression by siRNA further reduced Nrf2 nuclear translocation after TGF-β1 treatment in L02 and LX2 cells (Fig. 17G). On the contrary, Nrf2 nuclear expression was up-regulated by TRIM31 in a concentration-dependent manner (Fig. 17H). As expected, IHC ...
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... blotting assays confirmed the interaction between TRIM31 and Nrf2 in human hepatocytes (Fig. 17A and B). Additionally, the interaction between TRIM31 and Nrf2 in L02 cells was weakened upon TGF-β1 stimulation (Fig. 17C). The interaction between TRIM31 and Nrf2 was verified in human HSCs (Fig. 17D and E), and was receded under TGF-β1 stress (Fig. 17F). Moreover, knockdown of TRIM31 expression by siRNA further reduced Nrf2 nuclear translocation after TGF-β1 treatment in L02 and LX2 cells (Fig. 17G). On the contrary, Nrf2 nuclear expression was up-regulated by TRIM31 in a concentration-dependent manner (Fig. 17H). As expected, IHC staining showed that Mul-rescued expression of nuclear ...
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... TRIM31 and Nrf2 in L02 cells was weakened upon TGF-β1 stimulation (Fig. 17C). The interaction between TRIM31 and Nrf2 was verified in human HSCs (Fig. 17D and E), and was receded under TGF-β1 stress (Fig. 17F). Moreover, knockdown of TRIM31 expression by siRNA further reduced Nrf2 nuclear translocation after TGF-β1 treatment in L02 and LX2 cells (Fig. 17G). On the contrary, Nrf2 nuclear expression was up-regulated by TRIM31 in a concentration-dependent manner (Fig. 17H). As expected, IHC staining showed that Mul-rescued expression of nuclear Nrf2 in liver of CCl 4 -challenged mice was completely abrogated upon hepatic-specific TRIM31 knockout (Fig. 17I and J), which was validated by ...
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... was verified in human HSCs (Fig. 17D and E), and was receded under TGF-β1 stress (Fig. 17F). Moreover, knockdown of TRIM31 expression by siRNA further reduced Nrf2 nuclear translocation after TGF-β1 treatment in L02 and LX2 cells (Fig. 17G). On the contrary, Nrf2 nuclear expression was up-regulated by TRIM31 in a concentration-dependent manner (Fig. 17H). As expected, IHC staining showed that Mul-rescued expression of nuclear Nrf2 in liver of CCl 4 -challenged mice was completely abrogated upon hepatic-specific TRIM31 knockout (Fig. 17I and J), which was validated by western blotting assay (Fig. 17K), partially demonstrating the requirement of TRIM31 for Mul to improve Nrf2 signaling. ...
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... after TGF-β1 treatment in L02 and LX2 cells (Fig. 17G). On the contrary, Nrf2 nuclear expression was up-regulated by TRIM31 in a concentration-dependent manner (Fig. 17H). As expected, IHC staining showed that Mul-rescued expression of nuclear Nrf2 in liver of CCl 4 -challenged mice was completely abrogated upon hepatic-specific TRIM31 knockout (Fig. 17I and J), which was validated by western blotting assay (Fig. 17K), partially demonstrating the requirement of TRIM31 for Mul to improve Nrf2 signaling. Together, these findings suggested that there might be an interaction between TRIM31 and ...
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... contrary, Nrf2 nuclear expression was up-regulated by TRIM31 in a concentration-dependent manner (Fig. 17H). As expected, IHC staining showed that Mul-rescued expression of nuclear Nrf2 in liver of CCl 4 -challenged mice was completely abrogated upon hepatic-specific TRIM31 knockout (Fig. 17I and J), which was validated by western blotting assay (Fig. 17K), partially demonstrating the requirement of TRIM31 for Mul to improve Nrf2 signaling. Together, these findings suggested that there might be an interaction between TRIM31 and ...
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... eliminated upon TRIM31 HepcKO under fibrotic stress. Taken together, all our findings demonstrated that inflammation and oxidative stress in hepatocytes contributed to HSCs activation and collagen deposition, which could be mitigated by Mul through improving TRIM31-regulated Nrf2 signaling pathways, consequently ameliorating hepatic fibrosis (Fig. 18). Therefore, Mul may be a promising therapeutic strategy for the treatment of liver ...
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... of NLRP3 inflammasome can result in severe liver inflammation, hepatocyte pyroptotic cell death, and hepatic fibrosis in mice [45], whereas depressing NLRP3 inflammasome ameliorates liver fibrosis. For instance, in an animal model of non-alcoholic steatohepatitis (NASH), NLRP3 inflammasome activation was indispensable in fibrotic response, Fig. 18. Proposed mechanisms indicate the effects of mulberrin on liver fibrosis. In brief, under fibrotic stimuli, TRIM31-mediated activation of Nrf2 signaling pathway was restrained, resulting in inflammatory response and oxidative stress in hepatocytes, which contributed to the HSC activation and hepatic fibrosis. Notably, mulberrin ...
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... mice with hepatocyte-specific TRIM31 knockout, disclosing the necessity of TRIM31 for Mul to perform its anti-inflammatory capacities. Collectively, both our in vivo and in vitro findings demonstrated that Mul could restrain hepatic inflammation by suppressing NLRP3 and NF-κB signaling pathways through the improvement of TRIM31 signaling (Fig. ...
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... Intriguingly, we found that siNrf2 markedly diminished the effects of Mul against ROS production and oxidative stress in TGF-β1-treated L02 cells, and accelerated TGF-β1-induced oxidative damage in vitro. Herein, we concluded that Mul-suppressed oxidative stress in liver was Nrf2-dependent, contributing to the amelioration of hepatic injury (Fig. ...
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... ameliorated HSCs activation and collagen deposition both in CCl 4 -induced mouse models and TGF-β1-stimulated HSCs. Mul mainly mitigated inflammatory response and oxidative stress in hepatocytes through improving TRIM31/Nrf2 signaling pathway, contributing to the blockage of HSCs activation and ameliorating liver fibrosis consequently (Fig. 18). Therefore, Mul can be considered as a promising therapeutic strategy for liver fibrosis management through improving TRIM31/Nrf2 ...
Citations
... which in turn inhibits glucose production, attenuates hepatic fibrosis by reducing CTGFmediated deposition of collagen fibers, and attenuates the accumulation of pro-inflammatory factors, cytokines, and chemokines mediated by c-jun phosphorylation, thereby reducing the inflammatory response (26). Another research team discovered a natural compound 'mulberrin' in mulberry branches that can effectively target activation of TRIM31, reduce oxidative stress and liver inflammation, thereby alleviating NASH (27). However, instead of using the traditional RING structural domain, TRIM16 uses the B-box structural domain to perform its ubiquitination function. ...
Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases and is closely associated with metabolic abnormalities. The causes of NAFLD are exceedingly complicated, and it is known that a variety of signaling pathways, endoplasmic reticulum stress, and mitochondrial dysfunction play a role in the pathogenesis of NAFLD. Recent studies have shown that ubiquitination and deubiquitination are involved in the regulation of the NAFLD pathophysiology. Protein ubiquitination is a dynamic and diverse post-translational alteration that affects various cellular biological processes. Numerous disorders, including NAFLD, exhibit imbalances in ubiquitination and deubiquitination. To highlight the significance of this post-translational modification in the pathogenesis of NAFLD and to aid in the development of new therapeutic approaches for the disease, we will discuss the role of enzymes involved in the processes of ubiquitination and deubiquitination, specifically E3 ubiquitin ligases and deubiquitinating enzymes that are important in the regulation of NAFLD.
... Inhibiting HSC activation serves as a potential therapeutic approach for treating liver fibrosis. The intricate and diverse chemical structures of natural products have demonstrated significant potential for combating liver fibrosis, and these products are promising candidates for novel drug development [26][27][28][29][30]. ...
Background
Liver fibrosis is a representative scarring response that can ultimately lead to liver cancer. However, relevant antifibrotic drugs for the effective treatment of liver fibrosis in humans have not yet been identified. Chikusetsusaponin IVa (CS-IVa) is derived from natural products and exhibits multiple biological activities; however, its efficacy and potential mechanism of action against liver fibrosis remains unclear.
Purpose
This study aimed to examine the antifibrotic properties and potential mechanisms of action of CS-IVa.
Methods
We constructed two mature mouse models (CCl 4 challenge and bile duct ligation) to evaluate the antifibrotic properties of CS-IVa in vivo. Proteomics analysis and transforming growth factor β1 (TGF-β1)-activated LX-2 cells were used to elucidate the potential effects and mechanisms. Molecular docking, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA) were used to detect the affinity and binding between CS-IVa and its target.
Results
We found that CS-IVa significantly alleviated liver fibrosis and injury by downregulating yes-associated protein (YAP) and tafazzin (TAZ) expression. In an in vitro model, CS-IVa suppressed TGF-β1-induced hepatic stellate cell (HSC) activation, as well as the mRNA and protein expression of COL1A1, α-SMA, YAP, and TAZ. Moreover, specific knockdown or inhibition of YAP did not enhance the suppressive effect of CS-IVa on HSC activation or fibrosis-associated protein expression. Molecular docking, SPR, and CETSA showed that CS-IVa could directly bind to YAP.
Conclusion
These findings demonstrated that the administration of CS-IVa effectively alleviated liver fibrosis by suppressing the YAP/TAZ pathways. In addition, CS-IVa could directly bind to YAP and act as a YAP inhibitor.
... Ecological imbalances and disorders of intestinal microflora can lead to damage to the intestinal barrier and intestinal inflammation, which in turn can lead to intestinal mucosal damage (25). Chinese medicine can improve chronic liver disease through intestinal flora (26)(27)(28). We found that the traditional Chinese medicine FAE had a substantial influence on the diversity of intestinal flora in mice, and the number of OTUs in the FAE group differed greatly from that in the other two groups. ...
Objectives
To explore the mechanism underlying the effect of Fructus Akebiae (FAE) against hepatic fibrosis in mice through combined network pharmacology, liver metabolomics, and 16S rDNA analyses of the gut microbiota.
Methods
In this study, we randomly divided mice into the control, model, FAE high-dose, FAE medium-dose, and FAE low-dose groups to analyze the pathological changes in the hepatic fibrosis and levels of the α-SMA, collagen 1, Nuclear Factor Kappa B (NF-κ B), Toll Like Receptor 4 (TLR4). The gut microbiota was analyzed through 16S rDNA sequencing analysis of liver metabolites using liquid chromatography-mass spectrometry. Furthermore, network pharmacology was used to determine the specific molecular regulation mechanism of FAE in hepatic fibrosis treatment.
Results
FAE treatment markedly improved the pathological changes in the hepatic fibrosis. Analysis revealed that FAE administration reversed the carbon tetrachloride (CCl4)-induced dysbiosis by increasing the abundance of Akkermansia and reducing that of Cyanobacteria. Additionally, metabolomic analysis showed that FAE treatment reversed the CCl4-induced metabolic disorders by regulating amino and nucleotide sugar metabolism. Furthermore, correlation analysis showed that Akkermansia and Verrucomicobiota were closely related to D-tolasaccharide and maltotetraose saccharide. Moreover, network pharmacology indicated that FAE might regulate the signaling pathway through the JUN/CASP3/NOS3/PTGS2/HSP90AA1 during treatment.
Conclusion
FAE may be a promising treatment for hepatic fibrosis, and its protective effects are associated with improvements in the microbiome and metabolic disorders.
... Numerous studies have highlighted the critical role of Trim31 in the regulation of fibrosis. The overexpression of Trim31 has been shown to ameliorate the fibrotic process in the kidney [11,14] and liver [45]. However, it is still unclear whether Trim31 plays a role in the regulation of fibrosis in the heart. ...
Tripartite motif-containing protein 31 (Trim31) is known to be involved in various pathological conditions, including heart diseases. Nonetheless, its specific involvement in heart failure (HF) has yet to be determined. In this study, we examined the function and mechanism of Trim31 in HF by using mice with cardiac-specific knockout (cKO) of Trim31. The HF mouse model was induced via the subcutaneous injection of isoproterenol (ISO). We observed a decrease in Trim31 expression in the heart tissues of mice with HF. Compared with wild-type (WT) mice, Trim31 cKO mice presented more severe characteristics of HF, including worsened cardiac dysfunction, hypertrophy, and fibrosis. However, these symptoms in Trim31 cKO mice were significantly reversed when they received an intramyocardial injection of recombinant adeno-associated virus (AAV) expressing Trim31. Excessive activation of the NLRP3 inflammasome, manifested by increased levels of NLRP3, ASC, cleaved Caspase-1, cleaved GSDMD, IL-1β, and IL-18, was observed in Trim31 cKO mice with HF. However, Trim31 overexpression effectively reversed the NLRP3 inflammasome activation in Trim31 cKO mice with HF. Selective inhibition of the NLRP3 inflammasome with the NLRP3 inhibitor MCC950 effectively reversed the worsened cardiac dysfunction, hypertrophy, and fibrosis observed in Trim31 cKO mice with HF. Overall, the findings from this study reveal a crucial role of Trim31 in HF. Trim31 deficiency may contribute to the progression of HF by promoting cardiac hypertrophy, fibrosis, and inflammation by facilitating the activation of the NLRP3 inflammasome. Therefore, Trim31 may hold significant potential as a therapeutic target for the treatment of HF.
... Interestingly, the uncontrolled secretion of inflammatory factors and chemokines by macrophages are important driving force for HF formation 10 . In addition, inflammation-induced changes in the liver microenvironment lead to dysregulation of liver function, reducing the capacity for self-repair and accelerating HF progression 11 . ...
Kupffer cells (KCs), as residents and sentinels of the liver, are involved in the formation of hepatic fibrosis (HF). However, the biological functions of circular RNAs (circRNAs) in KCs to HF have not been determined. In this study, the expression levels of circRNAs, microRNAs, and messenger RNAs (mRNAs) in KCs from a mouse model of HF mice were investigated using microarray and circRNA-Seq analyses. circDcbld2 was identified as a candidate circRNA in HF, as evidenced by its up-regulation in KCs. Silver staining and mass spectrometry showed that Wtap and Igf2bp2 bind to cirDcbld2. The suppression of circDcbld2 expression decreased the KC inflammatory response and oxidative stress and inhibited hepatic stellate cell (HSCs) activation, attenuating mouse liver fibrogenesis. Mechanistically, Wtap mediated the N⁶-methyladenosine (m6A) methylation of circDcbld2, and Igf2bp2 recognized m6A-modified circDcbld2 and increased its stability. circDcbld2 contributes to the occurrence of HF by binding miR-144-3p/Et-1 to regulate the inflammatory response and oxidative stress. These findings indicate that circDcbld2 functions via the m6A/circDcbld2/miR-144-3p/Et-1 axis and may act as a potential biomarker for HF treatment.
... Two more recent studies have demonstrated that TRIM31 alleviates NAFLD and NASH pathologies by targeted degradation of rhomboid 5 homolog 2 (Rhbdf2) (35) and TAK1 (36) in the liver. TRIM31 is also responsible for the antifibrotic effects of mulberrin (a bioactive phytochemical from the traditional Chinese medicine Ramulus Mori) in CCl 4 -induced liver fibrosis (37). These lines of evidence illustrate the therapeutic potential of targeting TRIM family members in different stages of NAFLD. ...
Nonalcoholic fatty liver disease (NAFLD) encompasses a disease continuum from simple steatosis to nonalcoholic
steatohepatitis (NASH). However, there are currently no approved pharmacotherapies for NAFLD, although several drugs
are in advanced stages of clinical development. Because of the complex pathophysiology and heterogeneity of NAFLD,
the identification of potential therapeutic targets is clinically important. Here, we demonstrated that tripartite motif
56 (TRIM56) protein abundance was markedly downregulated in the livers of individuals with NAFLD and of mice fed
a high-fat diet. Hepatocyte-specific ablation of TRIM56 exacerbated the progression of NAFLD, while hepatic TRIM56
overexpression suppressed it. Integrative analyses of interactome and transcriptome profiling revealed a pivotal role of
TRIM56 in lipid metabolism and identified the lipogenesis factor fatty acid synthase (FASN) as a direct binding partner
of TRIM56. TRIM56 directly interacted with FASN and triggered its K48-linked ubiquitination–dependent degradation.
Finally, using artificial intelligence–based virtual screening, we discovered an orally bioavailable small-molecule inhibitor
of FASN (named FASstatin) that potentiates TRIM56-mediated FASN ubiquitination. Therapeutic administration of
FASstatin improved NAFLD and NASH pathologies in mice with an optimal safety, tolerability, and pharmacokinetics
profile. Our findings provide proof of concept that targeting the TRIM56/FASN axis in hepatocytes may offer potential
therapeutic avenues to treat NAFLD.
... Furthermore, the MAFLD therapeutic effects of SZ-A were believed to be associated with the activation of several substances as mentioned above. Moreover, relevant studies also suggested that SZ-A could activate the NRF2 axis to inhibit the excitation of hepatic stellate cells, thereby alleviating liver fibrosis [21,22]. It was also observed that PGC1α could regulate the expression of nuclear/mitochondrial genes associated with oxidative phosphorylation by coactivating NRF2, indicating that NRF2 may also act as an important pathway through which SZ-A exerts MAFLD-remedying effects [23]. ...
Background/Objectives: Metabolic-associated fatty liver disease (MAFLD) is one of the most common liver disorders associated with obesity and metabolic syndrome, and poses a significant global health burden with limited effective treatments. The aim of this study was to assess the protective effects of mulberry twig alkaloids (SZ-A) on MAFLD and to further investigate the underlying mechanisms including the specific targets or pathways. Methods: Diet-induced obesity (DIO) and normal mouse models were established by feeding C57Bl/6J mice with a high-fat diet (HFD) or common diet for 12 weeks. SZ-A, dapagliflozin, and placebo were administered to corresponding mouse groups for 8 weeks. Data of fasting blood glucose, glucose tolerance, insulin tolerance, and the body weight of mice were collected at the baseline and termination of the experiment. Serum liver enzymes and lipids were measured by ELISA. Western blotting, qPCR, and pathological section staining were implemented to evaluate the degrees of liver steatosis, fibrosis, and oxidative stress in mice. Results: In DIO mouse models, high-dose SZ-A (800 mg/kg/d) treatment significantly inhibited HFD-induced weight gain, improved insulin tolerance, and reduced serum alanine aminotransferase, total cholesterol, and triglyceride levels compared with placebo. In DIO mice, SZ-A could alleviate the pathological changes of hepatic steatosis and fibrosis compared with placebo. Lipid catabolism and antioxidant stress-related proteins were significantly increased in the livers of the high-dose SZ-A group (p < 0.05). Inhibition of PGC1α could inhibit the function of SZ-A to enhance lipid metabolism in hepatocytes. PGC1α might interact with NRF2 to exert MAFLD-remedying effects. Conclusions: By regulating the expression of PGC1α and its interacting KEAP1/NRF2 pathway in mouse liver cells, SZ-A played important roles in regulating lipid metabolism, inhibiting oxidative stress, and postponing liver fibrosis in mice with MAFLD.
... For IF analysis, the HET-1A cells after treatments were washed with PBS and were then blocked in 10% goat serum (#C0265, Beyotime Biotechnology) containing 0.3% Triton X-100 (#ST797, Beyotime Biotechnology) for 1 h at room temperature and incubated overnight with primary antibody NLRP3 (1:1000 #C1006, Beyotime Biotechnology). Photographs were captured using a fluorescence microscope [31]. ...
Background
Reflux esophagitis (RE) is a disease in which inflammation of the esophageal mucosa owing to the reflux of gastric contents into the esophagus results in cytokine damage. Britannilactone 1-O-acetate (Brt) has anti-inflammatory effects, significantly inhibiting the activation of the NLRP3 inflammasome, leading to a decrease in inflammatory factors including IL-1 β, IL-6, and TNF-α. However, the mechanism underlying its protective effect against RE-induced esophageal injury remains unclear. In the present study, we investigated the protective mechanism of TRIM31 against NLRP3 ubiquitination-induced RE both in vivo and in vitro.
Methods
A model of RE was established in vivo in rats by the method of “4.2 mm pyloric clamp + 2/3 fundoplication”. In vitro, the mod was constructed by using HET-1A (esophageal epithelial cells) and exposing the cells to acid, bile salts, and acidic bile salts. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was used to screen the concentration of administered drugs, and the viability of HET-1A cells in each group. HE staining was used to assess the degree of pathological damage in esophageal tissues. Toluidine blue staining was used to detect whether the protective function of the esophageal epithelial barrier was damaged and restored. The enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of IL-1 β, IL-6, and TNF-α factors in serum. Immunohistochemistry (IHC) was used to detect the expression level of NLRP3 in esophageal tissues. The molecular docking and Co-immunoprecipitation assay (Co-IP assay) were used to detect the TRIM31 interacts with NLRP3. Western blotting detected the Claudin-4, Claudin-5, The G-protein-coupled receptor calcium-sensitive receptor (CaSR), NLRP3, TRIM31, ASC, C-Caspase1, and Caspase1 protein expression levels.
Results
Brt could alleviate RE inflammatory responses by modulating serum levels of IL-1 β, IL-6, and TNF-α. It also activated the expression of NLRP3, ASC, Caspase 1, and C-Caspase-1 in HET-1A cells. Brt also attenuated TRIM31/NLRP3-induced pathological injury in rats with RE through a molecular mechanism consistent with the in vitro results.
Conclusions
Brt promotes the ubiquitination of NLRP3 through TRIM31 and attenuates esophageal epithelial damage induced by RE caused by acidic bile salt exposure. This study provides valuable insights into the mechanism of action of Brt in the treatment of RE and highlights its promising application in the prevention of NLRP3 inflammatory vesicle-associated inflammatory pathological injury.
Graphical Abstract
Supplementary Information
The online version contains supplementary material available at 10.1186/s13020-024-00986-y.
... The culture medium used was Dulbecco's Modified Eagle Medium supplemented with 10 % FBS and 1 % penicillin/streptomycin. Prior to intervention, LX2 was cultured in vitro and pre-treated 1,25(OH) 2 D 3 (100 nM) for 16 h [21,30]. Subsequently, TGF-β1 (10 ng/mL) was administered to induce LX2 activation for 12 h [31], followed by treatment with VP (10 nM) or siRNA in combination with 1,25 (OH) 2 D 3 (100 nM) for 12 h. Upon completion the experiment, protein and RNA were extracted from the cells and analyzed individually. ...
... Multiple cytokines induced the activation of NF-κB pathway, thus contributed to the enhance of inflammation response, EMT and tumorigenesis. Ge et al. found that the function of Mulberrin against hepatic fibrosis and oxidative stress was depended on the expression of TRIM31 and the suppression of NF-κB pathway and NOD-like receptor protein 3 (NLRP3) inflammasome [49]. In pancreatic cancer and colorectal cancer, TRIM31 played a role in the activation of the NF-κB and the downstream genes [41,50]. ...
Most TRIM family members characterized by the E3-ubiquitin ligases, participate in ubiquitination and tumorigenesis. While there is a dearth of a comprehensive investigation for the entire family in gastric cancer (GC). By combining the TCGA and GEO databases, common TRIM family members (TRIMs) were obtained to investigate gene expression, gene mutations, and clinical prognosis. On the basis of TRIMs, a consensus clustering analysis was conducted, and a risk assessment system and prognostic model were developed. Particularly, TRIM31 with clinical prognostic and diagnostic value was chosen for single-gene bioinformatics analysis, in vitro experimental validation, and immunohistochemical analysis of clinical tissue microarrays. The combined dataset consisted of 66 TRIMs, of which 52 were differentially expressed and 43 were differentially prognostic. Significant survival differences existed between the gene clusters obtained by consensus clustering analysis. Using 4 differentially expressed genes identified by multivariate Cox regression and LASSO regression, a risk scoring system was developed. Higher risk scores were associated with a poorer prognosis, suppressive immune cell infiltration, and drug resistance. Transcriptomic data and clinical sample tissue microarrays confirmed that TRIM31 was highly expressed in GC and associated with a poor prognosis. Pathway enrichment analysis, cell migration and colony formation assay, EdU assay, reactive oxygen species (ROS) assay, and mitochondrial membrane potential assay revealed that TRIM31 may be implicated in cell cycle regulation and oxidative stress-related pathways, contribute to gastric carcinogenesis. This study investigated the whole functional and expression profile and a risk score system based on the TRIM family in GC. Further investigation centered around TRIM31 offers insight into the underlying mechanisms of action exhibited by other members of its family in the context of GC.
Supplementary Information
The online version contains supplementary material available at 10.1186/s40246-024-00631-7.
