Effect of salvianolate on intestinal epithelium tight junction protein zonula occludens protein 1 in cirrhotic rats

AIM:To study the effect of salvianolate on tight junctions(TJs) and zonula occludens protein 1(ZO-1) in small intestinal mucosa of cirrhotic rats.METHODS:Cirrhosis was induced using carbon tetrachloride.Rats were randomly divided into the untreated group,low-dose salvianolate(12 mg/kg) treatment group,medium-dose salvianolate(24 mg/kg) treatment group,and high-dose salvianolate(48 mg/kg) treatment group,and were treated for 2 wk.Another 10 healthy rats served as the normal control group.Histological changes in liver tissue samples were observed under a light microscope.We evaluated morphologic indices of ileal mucosa including intestinal villi width and thickness of mucosa and intestinal wall using a pathological image analysis system.Ultrastructural changes in small intestinal mucosa were investigated in the five groups using transmission electron microscopy.The changes in ZO-1 expression,a tight junction protein,were analyzed by immunocytochemistry.The staining index was calculated as the product of the staining intensity score and the proportion of positive cells.RESULTS:In the untreated group,hepatocytes showed a disordered arrangement,fatty degeneration was extensive,swelling was obvious,and disorganized lobules were divided by collagen fibers in hepatic tissue,which were partly improved in the salvianolate treated groups.In the untreated group,abundant lymphocytes infiltrated the fibrous tissue with proliferation of bile ducts,and collagen fibers gradually decreased and damaged hepatic lobules were partly repaired following salvianolate treatment.Compared with the untreated group,no differences in intestinal villi width between the five groups were observed.The villi height as well as mucosa and intestinal wall thickness gradually thickened with salvianolate treatment and were significantly shorter in the untreated group compared with those in the salvianolate treatment groups and normal group(P < 0.01).The number of microvilli decreased and showed irregular lengths and arrangements in the untreated group.The intercellular space between epithelial cells was wider.The TJs were discontinuous,which indicated disruption in TJ morphology in the untreated group.In the treated groups,the microvilli in the intestinal epithelium were regular and the TJs were gradually integrated and distinct.The expression of ZO-1 decreased in the small intestine of the untreated cirrhotic rats.The high expression rate of ZO-1 in ileal mucosa in the untreated group was significantly lower than that in the medium-dose salvianolate group(21.43% vs 64.29%,χ 2 = 5.25,P < 0.05),high-dose salvianolate group(21.43% vs 76.92%,χ 2 = 8.315,P < 0.01) and normal group(21.43% vs 90%,χ 2 = 10.98,P < 0.01).CONCLUSION:Salvianolate improves liver histopathological changes,repairs intestinal mucosa and TJ structure,and enhances ZO-1 expression in the small intestinal mucosa in cirrhotic rats.

Nimbolide inhibits tumor growth by restoring hepatic tight junction protein expression and reduced inflammation in an experimental hepatocarcinogenesis

BACKGROUND Altered tight junction(TJ)proteins are correlated with carcinogenesis and tumor development.Nimbolide is a tetranotriterpenoid that has been shown to have antioxidant and anti-proliferative properties;however,its anticancer effects and molecular mechanism in hepatocellular carcinoma(HCC)remains obscure.AIM To investigate the effect of nimbolide on TJ proteins,cell cycle progression,and hepatic inflammation in a mouse model of HCC.METHODS HCC was induced in male Swiss albino mice(CD-1 strain)by a single intraperitoneal injection of 100 mg/kg diethylnitrosamine(DEN)followed by 80 ppm N-nitrosomorpholine(NMOR)in drinking water for 28 wk.After 28 wk,nimbolide(6 mg/kg)was given orally for four consecutive weeks in DEN/NMOR induced HCC mice.At the end of the 32nd week,all the mice were sacrificed and blood and liver samples were collected for various analyses.Macroscopic examinations of hepatic nodules were assessed.Liver histology and HCC tumor markers such as alpha-fetoprotein(AFP)and glypican-3 were measured.Expression of TJ proteins,cell proliferation,and cell cycle markers,inflammatory markers,and oxidative stress markers were analyzed.In silico analysis was performed to confirm the binding and modulatory effect of nimbolide on zonula occludens 1(ZO-1),nuclear factor of kappa light polypeptide gene enhancer in B-cells(NF-κB),and tumor necrosis factor alpha(TNF-α).RESULTS We found nimbolide treatment at a concentration of 6 mg/kg to HCC mice reduced hepatic tumor size by 52.08%and tumor volume(P<0.01),and delayed tumor growth in HCC mice with a concomitant reduction in tumor markers such as AFP levels(P<0.01)and glypican-3 expression(P<0.05).Furthermore,nimbolide treatment increased tight junction proteins such as ZO-1 and occludin expression(P<0.05,respectively)and reduced ZO-1 associated nucleic acid binding protein expression(P<0.001)in HCC mice liver.Nimbolide treatment to HCC mice also inhibited cell proliferation and suppressed cell cycle progression by attenuating proliferating cell nuclear antigen(P<0.01),cyclin dependent kinase(P<0.05),and CyclinD1(P<0.05)expression.In addition,nimbolide treatment to HCC mice ameliorated hepatic inflammation by reducing NF-κB,interleukin 1 beta and TNF-αexpression(P<0.05,respectively)and abrogated oxidative stress by attenuating 4-hydroxynonenal expression(P<0.01).Molecular docking studies further confirmed that nimbolide interacts with ZO-1,NF-κB,and TNF-α.CONCLUSION Our current study showed for the first time that nimbolide exhibits anticancer effect by reducing tumor size,tumor burden and by suppressing cell cycle progression in HCC mice.Furthermore,nimbolide treatment to HCC mice ameliorated inflammation and oxidative stress,and improved TJ proteins expression.Consequently,nimbolide could be potentially used as a natural therapeutic agent for HCC treatment,however further human studies are warranted.