Sai Zhu’s research while affiliated with Anhui Medical University and other places

Inflammation plays an essential role in eliminating microbial pathogens and repairing tissues, while sustained inflammation accelerates kidney damage and disease progression. Therefore, understanding the mechanisms of the inflammatory response is vital for developing therapies for inflammatory kidney diseases like acute kidney injury (AKI), which currently lacks effective treatment. Here, we identified N-acetyltransferase 10 ( NAT10 ) as an important regulator for acute inflammation. NAT10 , the only known “writer” protein for N4-acetylcytidine (ac4C) acetylation, is elevated in renal tubules across various AKI models, human biopsies, and cultured tubular epithelial cells (TECs). Conditional knockout (cKO) of NAT10 in mouse kidneys attenuates renal dysfunction, inflammation, and infiltration of macrophages and neutrophils, whereas its conditional knock-in (cKI) exacerbates these effects. Mechanistically, our findings from ac4C-RIP-seq and RNA-seq analyses revealed that NAT10-mediated ac4C acetylation enhances the mRNA stability of a range of key chemokines, including C-C motif chemokine ligand 2 ( CCL2 ) and C-X-C motif chemokine ligand 1( CXCL1 ), promoting macrophage and neutrophil recruitment and accelerating renal inflammation. Additionally, CCL2 and CXCL1 neutralizing antibodies or their receptor inhibitors, abrogated renal inflammation in NAT10 -overexpression TECs or NAT10 -cKI mice. Importantly, inhibiting NAT10 , either through Adeno-associated virus 9 (AAV9)-mediated silencing or pharmacologically with our found inhibitor Cpd-155, significantly reduces renal inflammation and injury. Thus, targeting the NAT10/CCL2/CXCL1 axis presents a promising therapeutic strategy for treating inflammatory kidney diseases.

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.

In hepatic fibrosis (HF), hepatic stellate cells (HSCs) form the extracellular matrix (ECM), and the pathological accumulation of ECM in the liver leads to inflammation. Our previous research found that miR-324-3p was down-regulated in culture-activated human HSCs. However, the precise effect of miR-324-3p on HF has not been elucidated. In this study, the HF mouse models were induced through directly injecting carbon tetrachloride (CCl4) into mice; the HF cell models were constructed using TGF-β1-treated LX-2 cells. Next, real-time-quantitative polymerase chain reaction (RT-qPCR), western blot (WB) and immunohistochemistry (IHC) were applied to assess the expression levels of miR-324-3p, α-smooth muscle actin (α-SMA), Vimentin or SMAD4; hematoxylin and eosin (H&E), Masson’ s trichrome and Sirius red staining to evaluate the liver injury; luciferase reporter assay to verify the targeting relationship between miR-324-3p and SMAD4; enzyme-linked immunosorbent assay (ELISA) to determine the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and cell counting kit-8 (CCK-8) and flow cytometry to evaluate the effects of miR-324-3p on cell proliferation and cycle/apoptosis, respectively. The experimental results showed a reduction in miR-324-3p level in CCl4-induced HF mice as well as transforming growth factor (TGF)-β1-activated HSCs. Interestingly, the miR-324-3p level was rescued following the HF recovery process. In HF mice induced by CCl4, miR-324-3p overexpression inhibited liver tissue damage, decreased serum ALT and AST levels, and inhibited fibrosis-related biomarkers (α-SMA, Vimentin) expression, thereby inhibiting HF. Similarly, miR-324-3p overexpression up-regulated α-SMA and Vimentin levels in HF cells, while knockdown of miR-324-3p had the opposite effect. Besides, miR-324-3p played an antifibrotic role through inhibiting the proliferation of hepatocytes. Further experiments confirmed that miR-324-3p targeted and down-regulated SMAD4 expression. SMAD4 was highly expressed in HF cells, and silencing SMAD4 significantly decreased the α-SMA and Vimentin levels in HF cells. Collectively, the miR-324-3p may suppress the activation of HSCs and HF by targeting SMAD4. Therefore, miR-324-3p is identified as a potential and novel therapeutic target for HF.

Acute liver injury (ALI) is a complex, life-threatening inflammatory liver disease, and persistent liver damage leads to rapid decline and even failure of liver function. However, the pathogenesis of ALI is still not fully understood, and no effective treatment has been discovered. Recent evidence shows that many circular RNAs (circRNAs) are associated with the occurrence of liver diseases. In this study we investigated the mechanisms of occurrence and development of ALI in lipopolysaccharide (LPS)-induced ALI mice. We found that expression of the circular RNA circDcbld2 was significantly elevated in the liver tissues of ALI mice and LPS-treated RAW264.7 cells. Knockdown of circDcbld2 markedly alleviates LPS-induced inflammatory responses in ALI mice and RAW264.7 cells. We designed and synthesized a series of hesperidin derivatives for circDcbld2, and found that hesperetin derivative 2a (HD-2a) at the concentrations of 2, 4, 8 μM effectively inhibited circDcbld2 expression in RAW264.7 cells. Administration of HD-2a (50, 100, 200 mg/kg. i.g., once 24 h in advance) effectively relieved LPS-induced liver dysfunction and inflammatory responses. RNA sequencing analysis revealed that the anti-inflammatory and hepatoprotective effects of HD-2a were mediated through downregulating circDcbld2 and suppressing the JAK2/STAT3 pathway. We conclude that HD-2a downregulates circDcbld2 to inhibit the JAK2/STAT3 pathway, thereby inhibiting the inflammatory responses in ALI. The results suggest that circDcbld2 may be a potential target for the prevention and treatment of ALI, and HD-2a may have potential as a drug for the treatment of ALI.

Circular RNA (circRNA) has been implicated in liver fibrosis and modulated by multiple elusive molecular mechanisms, while the effects of N6-methyladenosine (m6A) modification on circRNA are still elusive. Herein, we identify circIRF2 from our circRNA sequencing data, which decreased in liver fibrogenesis stage and restored in resolution stage, indicating that dysregulated circIRF2 may be closely associated with liver fibrosis. Gain/loss-of-function analysis was performed to evaluate the effects of circIRF2 on liver fibrosis at both the fibrogenesis and resolution in vivo. Ectopic expression of circIRF2 attenuated liver fibrogenesis and HSCs activation at the fibrogenesis stage, whereas downregulation of circIRF2 impaired mouse liver injury repair and inflammation resolution. Mechanistically, YTHDF2 recognized m6A-modified circIRF2 and diminished circIRF2 stability, partly accounting for the decreased circIRF2 in liver fibrosis. Microarray was applied to investigate miRNAs regulated by circIRF2, our data elucidate cytoplasmic circIRF2 may directly harbor miR-29b-1-5p and competitively relieve its inhibitory effect on FOXO3, inducing FOXO3 nuclear translocation and accumulation. Clinically, circIRF2 downregulation was prevalent in liver fibrosis patients compared with healthy individuals. In summary, our findings offer a novel insight into m6A modification-mediated regulation of circRNA and suggest that circIRF2 may be an exploitable prognostic marker and/or therapeutic target for liver fibrosis.

N6-methyladenosine (m6A) is a dynamic and reversible epigenetic regulation. As the most prevalent internal post-transcriptional modification in eukaryotic RNA, it participates in the regulation of gene expression through various mechanisms, such as mRNA splicing, nuclear export, localization, translation efficiency, mRNA stability, and structural transformation. The involvement of m6A in the regulation of gene expression depends on the specific recognition of m6A-modified RNA by reader proteins. In the pathogenesis and treatment of liver disease, studies have found that the expression levels of key genes that promote or inhibit the development of liver disease are regulated by m6A modification, in which abnormal expression of reader proteins determines the fate of these gene transcripts. In this review, we introduce m6A readers, summarize the recognition and regulatory mechanisms of m6A readers on mRNA, and focus on the biological functions and mechanisms of m6A readers in liver cancer, viral hepatitis, non-alcoholic fatty liver disease (NAFLD), hepatic fibrosis (HF), acute liver injury (ALI), and other liver diseases. This information is expected to be of high value to researchers deciphering the links between m6A readers and human liver diseases.

Hepatic fibrosis (HF) is a reversible wound-healing response characterized by excessive extracellular matrix (ECM) deposition and secondary to persistent chronic injury. Bromodomain protein 4 (BRD4) commonly functions as a "reader" to regulate epigenetic modifications involved in various biological and pathological events, but the mechanism of HF remains unclear. In this study, we established a CCl4-induced HF model and spontaneous recovery model in mice and found aberrant BRD4 expression, which was consistent with the results in human hepatic stellate cells (HSCs)- LX2 cells in vitro. Subsequently, we found that distriction and inhibition of BRD4 restrained TGFβ-induced trans-differentiation of LX2 cells into activated, proliferative myofibroblasts and accelerated apoptosis, and BRD4 overexpression blocked MDI-induced LX2 cells inactivation and promoted the proliferation and inhibited apoptosis of inactivated cells. Additionally, adeno-associated virus serotype 8-loaded short hairpin RNA-mediated BRD4 knockdown in mice significantly attenuated CCl4-induced fibrotic responses including HSCs activation and collagen deposition. Mechanistically, BRD4 deficiency inhibited PLK1 expression in activated LX2 cells, and ChIP and Co-IP assays revealed that BRD4 regulation of PLK1 was dependent on P300-mediated acetylation modification for H3K27 on the PLK1 promoter. In conclusion, BRD4 deficiency in the liver alleviates CCl4-induced HF in mice, and BRD4 participates in the activation and reversal of HSCs through positively regulating the P300/H3K27ac/PLK1 axis, providing a potential insight for HF therapy.

Online reviews often contain considerable information redundancy. Understanding how to mine and identify valuable information is an important research topic. Based on knowledge-based theory and social network theory, this study discusses the impact of knowledge redundancy in online travel reviews on consumer purchase behaviour from the perspective of knowledge networks. By crawling 943,516 online reviews of 609 tourism products from Trip and Tuniu, we conduct an empirical analysis of the relationships among knowledge redundancy, knowledge diversity and consumer purchase behaviour. The results show that there is a U-shaped relationship between knowledge redundancy in online tourism reviews and consumer purchase behaviour, and knowledge diversity plays an intermediary role in the relationship between knowledge redundancy and consumer purchase behaviour. The results provide valuable and timely insights for the development of the online tourism industry.

Acute kidney injury (AKI) is a clinical syndrome that is defined as a sudden decline in renal function and characterized by inflammation and programmed cell death of renal tubular epithelial cells. Necroptosis is a form of regulated cell death that requires activation of receptor interacting protein kinase 3 (RIPK3) and its phosphorylation of the substrate MLKL. RIPK3 plays an important role in acute kidney injury, and hence developing its inhibitors is considered as one of the promising strategies aimed at prevention and treatment of AKI. Recently, we discovered AZD5423 as a novel potent RIPK3 inhibitor using a computer-aided hybrid virtual screening strategy according to three-dimensional structure of RIPK3. Our findings revealed that AZD5423 strongly inhibits activation of RIPK3, and MLKL phosphorylation upon cisplatin-, hypoxia/reoxygenation (H/R)- and TNF-α stimuli as compared with GSK872, which is a previously identified RIPK3 inhibitor. Importantly, AZD5423 exerts effective protection against cisplatin- and ischemia/reperfusion (I/R)-induced AKI mouse model. The results of cellular thermal shift assay and experiments in RIPK3 knockout cells indicated that AZD5423 could directly target RIPK3 to inhibit RIPK3 kinase activity. Mechanistically, the docking of AZD5423 and RIPK3 suggested that the kinase domain of RIPK3 for Lys⁵⁰, Arg³¹³, Lys²⁹, Arg³⁷ might form hydrogen bonds with AZD5423. Site-directed mutagenesis further revealed that AZD5423 reduces injury response via interacting with the key RIPK3 amino acid residues of Lys⁵⁰ and Arg³¹³. In conclusion, our study has demonstrated that AZD5423 may serve as a potent inhibitor of RIPK3 kinase and a promising clinical candidate for AKI treatment.