INDO-GLOBAL RESEARCH LIBRARY EVALUATION OF EFFECT OF STRESS ON CHOLESTEROL AND INTESTINE OF MICE

Abstract

Objective: Stress is now a day's part of our lifestyle. Every individual faces stress in their day to day activity. When humans experience stress, as a survival mechanism, the body diverts energy, blood, enzymes and oxygen from the digestive organs to other areas of the body. In addition to mental and emotional stresses like fear, anger and worry, physical stresses including infections, trauma from injuries, surgery and environmental toxins can have a major effect on our digestive efficiency. In stress, proteins from the thymus and lymph glands are broken down into sugar for immediate energy. Also fat is mobilized from storage depots. Cortisol released in response to stress stimulates gastric-acid secretion. Methods: Mice were randomly divided into three groups (n=10 in each). One group served as control and epinephrine was administered intramuscularly to other two groups at the dose of 100nl/kg b.w. and 200nl/kg b.w. respectively. Animals were sacrificed after two weeks and four weeks of treatment, blood was collected for biochemical assay and tissues were fixed for histological study. Results: Cholesterol level and SGPT level in mice were increased after increased amount of stress. Stress also causes degeneration in muscular layer of intestine which adversely affects peristaltic movement of intestine. Goblet cells and endothelial cells were also degenerated leading to improper absorption of food. As level and duration of stress increases degeneration is also increases. Conclusion: Thus it is evident from study that stress adversely affects digestion and absorption of food in intestine and increased cholesterol may lead to cardiac arrest. The significant finding (s) of the study: Stress causes degeneration of villi in intestine due to increased epinephrine amount which finally causes improper absorption of digested food leading malnutrition in mice. Cholesterol and SGPT level were also increased after stress. This study adds: This is helpful to find digestive anomalies in stressed people, increased cholesterol was also related with increased number of heart patients now a days. If general individual know the role of stress and digestive system they may either take least stress or practice some anti stress therapy, which is helpful for them.

Full text

289
INDO-GLOBAL RESEARCH LIBRARY
www.igrlinfo.com
INDO-GLOBAL RESEARCH LIBRARY
Indo-Global Research Journal of Pharmaceutical Sciences
April-June 2012 | Vol. 2 | Issue 2
Available online www.igrlinfo.com
EVALUATION OF EFFECT OF STRESS ON CHOLESTEROL AND INTESTINE OF MICE
KUMAR RANJIT *, ANSHUPRIYA, KUMAR ARUN, SINGH J.K., NATH A. AND ALI MOHAMMAD
Mahavir Cancer Institute & Research Centre, Phulwarisharif, Patna (Bihar), India
E-mail– ranjitzool17@gmail.com
ABSTRACT
Objective: Stress is now a day’s part of our lifestyle. Every individual faces stress in their day to day activity. When humans experience stress, as a
survival mechanism, the body diverts energy, blood, enzymes and oxygen from the digestive organs to other areas of the body. In addition to mental
and emotional stresses like fear, anger and worry, physical stresses including infections, trauma from injuries, surgery and environmental toxins
can have a major effect on our digestive efficiency. In stress, proteins from the thymus and lymph glands are broken down into sugar for immediate
energy. Also fat is mobilized from storage depots. Cortisol released in response to stress stimulates gastric-acid secretion. Methods: Mice were
randomly divided into three groups (n=10 in each). One group served as control and epinephrine was administered intramuscularly to other two
groups at the dose of 100nl/kg b.w. and 200nl/kg b.w. respectively. Animals were sacrificed after two weeks and four weeks of treatment, blood
was collected for biochemical assay and tissues were fixed for histological study. Results: Cholesterol level and SGPT level in mice were increased
after increased amount of stress. Stress also causes degeneration in muscular layer of intestine which adversely affects peristaltic movement of
intestine. Goblet cells and endothelial cells were also degenerated leading to improper absorption of food. As level and duration of stress increases
degeneration is also increases. Conclusion: Thus it is evident from study that stress adversely affects digestion and absorption of food in intestine
and increased cholesterol may lead to cardiac arrest. The significant finding (s) of the study: Stress causes degeneration of villi in intestine due to
increased epinephrine amount which finally causes improper absorption of digested food leading malnutrition in mice. Cholesterol and SGPT level
were also increased after stress. This study adds: This is helpful to find digestive anomalies in stressed people, increased cholesterol was also
related with increased number of heart patients now a days. If general individual know the role of stress and digestive system they may either take
least stress or practice some anti stress therapy, which is helpful for them.
Key words: Stress, epinephrine, goblet cells, cholesterol and SGPT.
INTRODUCTION
Stress, defined as an acute threat to the homeostasis of an
organism,
1-3
be the real (physical) or perceived (psychological), and
whether posed by events in the outside world or from within,
evokes adaptive responses which serve to defend the stability of the
internal environment and to assure the survival of the organism.
4-6
Now-a-days each and every people are suffering from a number of
genetic as well as physiological disorders that are caused by long-
term exposure to stress including hypertension, depression, high
blood pressure, heart attack, cancer, asthma, obesity, infertility etc.
Stress can manifest itself from several sources, be it bodily stress
from sickness, injury, dehydration or poor nutrition, social stress
from unpleasant events or disagreeable altercations with others,
psychological stress from depression, worry, low self esteem or a
lifestyle that includes alcohol or drug abuse and work related stress.
Whatever influence causes our specific stress, it is invariably
expressed in some form of physical or mental symptoms. Health care
experts estimate that 75-90 percent of all visits to primary care
physicians are due to stress related problems.
The secretion of stress hormones (glucagon, catecholamines, cortisol
and GH) and especially cortisol increases during the acute stress and
emotional stimuli.
7-12
Prolonged cortisol secretion (which may be
due to chronic stress or the excessive secretion seen in Cushing's
syndrome) results in significant physiological changes.
13
Cortisol
counteracts insulin, contributes to hyperglycemia-causing
hepatic gluconeogenesis and inhibits the peripheral utilization of
glucose (insulin resistance) by decreasing the translocation
of glucose transporters (especially GLUT4) to the cell
membrane.
14,15
However, cortisol increases glycogen synthesis
(glycogenesis) in the liver.
16
Cortisol's only direct effect on the
hydrogen ion excretion of the kidneys is to stimulate the excretion of
ammonium ions by deactivating the renal glutaminase enzyme.
17
Thus the present work is designed to find out effect of stress on
SGPT level, Cholesterol level and histology of intestine of mice.
MATERIALS AND METHODS
1. Chemical: Epinephrine was used.
2. Experimental model: Reared sexually matured 6-8 weeks old
age group male and female Swiss Albino mice (Mus musculus)
weighing 25-35gm b.w. in the animal house section of Mahavir
Cancer Institute and Research Centre, Patna, were selected as an
experimental model in the present study. The animals were housed
at controlled environmental conditions 22±2ºC, relative humidity
50±10%, and 12h dark-light cycle. Animals were housed and
allowed to free access to food and water. All experimental
procedures were conducted as per the guidelines of CPCSEA
(Committee for the Purpose of Control and Supervision of
Experiments on Animals).
3. Methodology:
a) Experimental protocol: Selected pathogen-free mice were
randomly divided into three groups (n=10 in each). One group
Research Article ISSN: 2249-4189

290
INDO-GLOBAL RESEARCH LIBRARY
www.igrlinfo.com
served as control and epinephrine was administered
intramuscularly to other two groups at the dose of 100nl/kg b.w.
and 200nl/kg b.w. respectively. Animals were sacrificed after two
weeks and four weeks of treatment with epinephrine in each group.
b) Histopathological Studies: The intestine was dissected out and
fixed in 10% neutral formalin solution and the tissue was processed.
The slides were stained with Haematoxylene and Eosin and
examined morphometrical under LM.
c) Biochemical Assessment: With the separated serum cholesterol
analysis and S.G.P.T. analysis were performed with standard kit
(Coral) to establish the effects of epinephrine induced stress.
RESULTS
Cholesterol level in control group was 130 mg/dl. It was 144 mg/dl
and 181 mg/dl after 2 weeks and 4 weeks of epinephrine 100 nl/kg
b.w administration. While it were 162 mg/dl and 195 mg/dl after 2
weeks and 4 weeks of epinephrine 200 nl/kg b.w administration
(Graph-1). SGPT level in control group was 18 IU/ml. It was 53
IU/ml and 82 IU/ml after 2 weeks and 4 weeks of epinephrine 100
nl/kg b.w administration. While it were 66 IU/ml and 109 IU/ml
after 2 weeks and 4 weeks of epinephrine 200 nl/kg b.w
administration (Graph-2).
Control group of intestine shows normal villi with well organized
circular and longitudinal muscles i.e. muscularis externa. Distinct
mucosa and sub-mucosa layers are also seen (Figure:1). Epinephrine
100 nl/kg b.w adminiatered for two weeks shows villi with
degenerated goblet cells. Lumen is also not distinctly visible
(Figure:2). Muscularis externa showing degeneration with some
vacuolated spaces in submucosa layer. Villi also showing
degeneration (Figure: 3). Epinephrine 100 nl/kg b.w adminiatered
for four weeks shows degenerated muscularis externa with
vacuolization in submucosa layer. Fragmented villi were also seen
(Figure:4). Epinephrine 100 nl/kg b.w adminiatered for four weeks
shows enlarged view of villi with clustered nuclei. Degenerated
muscularis externa were also seen (Figure:5). Epinephrine 200
nl/kg b.w adminiatered for two weeks shows rudimentary
muscularis externa with many vacuolated spaces. Villi with
clustered nuclei were seen with many vacuolated spaces in
submucosa layer (Figure: 6).
Epinephrine 200 nl/kg b.w adminiatered for two weeks shows
enlarged view of degenerated muscularis externa with vacuolization
in submucosa layer (Figure:7). Epinephrine 200 nl/kg b.w
adminiatered for four weeks shows complete fragmentation of villi
with clustered nuclei. Goblet cells were also degenerated (Figure:8).
Epinephrine 200 nl/kg b.w adminiatered for four weeks shows
enlarged view of muscularis externa with complete rupture of
longitudinal muscle. Villi with complete degeneration of goblet cells
were also seen. Degenerated cytoplasm and lacteals were also
prominent (Figure: 9).
DISCUSSION
The association between psychological stress and small intestinal
motility has been postulated for about 20 years. Some studies in
experimental animals indicated contradictory results, which may be
due to different stressors in part. Varied stressors can influence
small intestinal motility via different mechanisms. Muelas et al.
18
demonstrated that restraint stress increased small intestinal
motility both during fasting and after food.
Ditto et al.
19
reported that a prolonged active coping stressor with
minimal motor requirements enhanced small intestinal transit.
However, Tsukada et al.
20-22
demonstrated that the small intestinal
transit was significantly inhibited by restraint stress but not by
footshock stress. And footshock stimulus may cancel the inhibition
of small intestinal motility by restraint stress. Leveau et al.
23
demonstrated that impairment in intestinal motility probably played
a pathophysiological role in the development of bacterial
overgrowth. We also find increased cholesterol in stressed mice, as
level of stress increases cholesterol concentration in serum were
also increases. Cortisol released in response to stress stimulates
gastric-acid secretion. Net chloride secretion in the intestines is
inversely decreased by cortisol in vitro (methylprednisolone).
24
Cortisol inhibits sodium loss through the small intestine of
mammals.
25
It also reduces calcium absorption in the intestine.
26
in
the study Serum Glutinine Pyruvate Test (SGPT) level were also
increased with increased amount of stress.
Beaumont
27
reported that normal gut function was disturbed in a
patient with a gastric fistula during periods of emotional stress.
Selye introduced stress research in animal models and characterized
induction of gastric ulcer as a classical response to stress
28
. In
present study degeneration were observed in villi prominently.
Mucosa and sub mucosa were rudimentary. Degeneration was also
prominent in muscular layer of intestine with distinct vacuolization
which finally causes improper peristaltic movement of intestine
leading to indigestion of food as well as reduced hunger.
Degenerated goblet cells were also prominent. Endothelial cells of
intestine were also degenerated which adversely affect absorption
in intestine. Clustered nuclei were also observed causes indigestion.
Increased stress causes increased level of glucose while lipid
peroxidation level also increases that leads to diabetes in mice.
29
Thus it is concluded from study that stress causes increase in
cholesterol level and SGPT level. Increased cholesterol with
increased amount of stress may lead to cardiac arrest. It also causes
degeneration in muscular layer of intestine which adversely affect
peristaltic movement of intestine due to which food movement
inside intestine is not properly occur, it causes indigestion of food.
Mucosa and sub-mucosa layer were also degenerated which
adversely affect assimilation of food. Goblet cells and endothelial
cells were also degenerated leading to improper absorption of food.
As level and duration of stress increases degeneration is also
increases. Thus it is evident from study that stress adversely affects
digestion and absorption of food in intestine.
ACKNOWLEDGEMENT
The authors are grateful to Mahavir Cancer Institute and Research
Centre, Patna for providing research facilities and we are also
thankful to all research laboratory staff and animal house staff for
their proper support during study.
REFERENCES
1. Selye H. Stress. Quebec: Acta Medical Publisher, 1950.
2. Chrousos GP, Gold PW. The concepts of stress and stress
system disorders: overview of physical and behavioral
homeostasis. JAMA 1992; 267:1244–52.
3. Chrousos GP, Loriaux DL, Gold PW. The concept of stress and
its historical development. Adv Exp Med Biol 1988; 245:3–7.
4. Sawchenko PE, Li H-Y. Circuits and mechanisms governing
hypothalamic responses to stress: a tale of two paradigms. In:
Ericsson A, Mayer EA, Saper CB, et al, eds. The biological basis
for mind body interactions, vol. 6, 122 edn. Amsterdam:
Elsevier Science, 2000:59–75.
5. Mayer EA. The neurobiology of stress and gastrointestinal
disease. Gut 2000; 47: 861–869.
6. Mayer EA., Bruce D. Naliboff, Lin Chang, and Santosh V.
Coutinho. Stress and the Gastrointestinal Tract. V. Stress and
irritable bowel syndrome. Am J Physiol Gastrointest Liver
Physiol 2001; 280: G519–G524.
7. Maryam Radahmadi et al; Effects of stress on exacerbation of
diabetes mellitus, serum glucose and cortisol levels and body
weights in rats; Pathophysiology 2006, 13: 51-55
8. Goldstein R.E., Cherrington A.D., Reed G.W., Lacy D.B.,
Wasserman D.H., Abumrad N.N.; Effects of chronic
hypercortisolemia on carbohydrate metabolism during
insulin deficiency; Am. J. Physiol. 1994, 266: E618-E627.

291
INDO-GLOBAL RESEARCH LIBRARY
www.igrlinfo.com
9. Schade D.S.and Eaton R.P.; The temporal relationship between
endogenously secreted stress hormones and metabolic
decompensation in diabetic man; J. Clin. Endocrinol. Metabol.
1980, 50(1): 131-136.
10. Wortsman J., Frank S., Cryer P.E.; Adrenomedullary response
to maximal stress in human; Am. J. Med. 1984, 77: 779-784.
11. Garces L.Y., Kenny F.M., Drash A., Preeyasombat C.; Cortisol
secretion in acidotic and non-acidotic juvenile diabetes
mellitus; J. Pediatr. 1969, 74(4): 517-522.
12. Barnes R.F., Raskind M., Gumbrecht G., Halter J.B.; The effects
of age on the plasma catecholamine response to mental stress
in man; J. Clin. Endocrinol. Metabol. 1982, 54(1): 64-69.
13. Scott, Elizabeth; "Cortisol and Stress: How Cortisol Affects
Your Body and How To Stay Healthy in the Face of Stress".
Stress.about.com. Retrieved 29 November 2011.
14. King, Michael W.; Lange Q&A USMLE Step 1(Sixth ed.). New
York: McGraw-Hill, Medical Pub. Division. 2005, p. 82.
15. Piroli, G. G.; Grillo, C. A.; Reznikov, L. R.; Adams, S.; McEwen, B.
S.; Charron, M. J.; Reagan, L. P.. "Corticosterone Impairs
Insulin-Stimulated Translocation of GLUT4 in the Rat
Hippocampus". Neuroendocrinology 2007, 85 (2): 71–80.
16. Baynes, J., Dominiczak, M., Medical Biochemistry. Elsevier
Limited; Third Edition (2009).
17. Kokshchuk, G.I.; Pakhmurnyi, B.A.; “Role of Glucocorticoids in
Regulation of the Acid-Excreting Function of the Kidneys”.
Fiziol. Z H SSR I.M.I.M. Sechenova 1979, 65: 751.
18. Muelas MS, Ramirez P, Parrilla P, Ruiz JM, Perez JM, Candel
MF, Aguilar J, Carrasco L. Vagal system involvement in
changes in small bowel motility during restraint stress: an
experimental study in the dog. Br J Surg 1993; 80: 479-483
19. Ditto B, Miller SB, Barr RG. A one-hour active coping stressor
reduces small bowel transit time in healthy young adults.
Psychosom Med 1998; 60: 7-10
20. Tsukada F, Sawamura K, Kohno H, Ohkubo Y. Mechanism of
inhibition of small intestinal motility by restraint stress
differs from that with norepinephrine treatment in rats. Biol
Pharm Bull 2002; 25: 122-124
21. Tsukada F, Sugawara M, Kohno H, Ohkubo Y. Evaluation of the
effects of restraint and footshock stress on small intestinal
motility by an improved method using a radionuclide, 51Cr, in
the rat. Biol Pharm Bull 2001; 24: 488-490
22. Tsukada F, Ohuchi Y, Terunuma T, Sugawara M, Kohno H,
Ohkubo Y. Activation of mu-opioid pathway is associated with
the canceling effect of footshock stimulus on the restraint
stress-induced inhibition of small intestinal motility in rats.
Biol Pharm Bull 2001; 24: 1332-1334
23. Leveau P, Wang X, Soltesz V, Ihse I, Andersson R. Alterations
in intestinal motility and microflora in experimental acute
pancreatitis. Int J Pancreatol 1996; 20: 119-125
24. Tai YH, Decker RA, Marnane WG, Charney AN, Donowitz M;
"Effects of methylprednisolone on electrolyte transport by in
vitro rat ileum". Am. J. Physiol. May 1981, 240 (5): G365–70.
25. Sandle GI, Keir MJ, Record CO; "The effect of hydrocortisone
on the transport of water, sodium, and glucose in the jejunum.
Perfusion studies in normal subjects and patients with coeliac
disease". Scand. J. Gastroenterol. 1981, 16 (5): 667–71.
26. Shultz TD, Bollman S, Kumar R; "Decreased intestinal calcium
absorption in vivo and normal brush border membrane
vesicle calcium uptake in cortisol-treated chickens: evidence
for dissociation of calcium absorption from brush border
vesicle uptake".Proc. Natl. Acad. Sci. U.S.A. June 1982, 79 (11):
3542–6.
27. Beaumont, W. Experiments and Observations on the Gastric
Juices and the PhysioZogy of Digestion. Edinburgh, UK: Neill,
1838.
28. Selye, H. The general adaptation syndrome and the diseases of
adaptation. J. CZin. EndocrinoZ. 6: 117-230, 1946.
29. Ranjit Kumar, Anshupriya, Arun Kumar, Md. Ali, A. Nath and
J.K. Singh; Increased epinephrine concentration during stress
in relation to diabetes in mice; Elixir Hor. & Sig. 36 (2011)
3058-3061.
Graph: 1
Graph: 2
Figure: 1
Control group of intestine showing T.S. of
normal villi with well organized circular and
longitudinal muscles i.e. muscularis externa.
Distinct mucosa and sub-mucosa layers are also
seen
.
Figure: 2
Epinephrine 100nl/kg b.w for 2 weeks
administered group shows villi with degenerated
goblet cells. Lumen was also n
ot distinctly visible.

292
INDO-GLOBAL RESEARCH LIBRARY
www.igrlinfo.com
Figure: 3
Epinephrine 100nl/kg b.w for 2 weeks
administered group shows degeneration with
some vacuolated spaces in submucosa layer. Villi
also show degeneration.
Figure: 4
Epinephrine 100nl/kg b.w for 4 weeks
administered group shows degenerated
muscularis externa with vacuolization in
submucosa layer. Fragmented villi were also
seen.
Figure: 5,
Epinephrine 100nl/kg b.w for 4
weeks administered group shows enlarged
view of villi with clustered nuclei.
Degenerated muscularis externa were also
seen.
Figure: 6,
Epinephrine 200nl/kg b.w for 2 weeks
administered group shows rudimentary
muscularis externa. Villi with clustered nuclei
were seen with degenerated submucosa layer.
Figure: 7,
Epinephrine 200nl/kg b.w for 2
weeks administered group shows enlarged
view of degenerated muscularis externa with
vacuolization in submucosa layer.
Figure: 8,
Epinephrine 200nl/kg b.w for 4
weeks administered group shows complete
fragmentation of villi with clustered nuclei.
Goblet cells were also degenerated.

293
INDO-GLOBAL RESEARCH LIBRARY
www.igrlinfo.com
Figure: 9
Epinephrine 200nl/kg b.w
for 4
weeks administered group shows complete
rupture of longitudinal muscle. Villi with
complete degeneration of goblet cells were
also seen. Degenerated cytoplasm and lacteals
were also prominent.
Date of Submission: 15/1/12
Date of Acceptance: 15/3/12
Conflict of Interest: Nil
Source of Support: Nil