Study On The Protective Role And Relative Mechanisms Of IPLA₂β In ConA-and DSS-induced Immunological Liver And Intestinal Injury

With the increasing industrial pollution and changing lifestyle, there are more and more xenobiotics in the environment which contributes to the immunological liver and intestinal injury. Hepatitis virus, drugs and alcohol can cause varying degrees of immunological liver injury;Heavy metals, formaldehyde and food antigens can induce intestinal inflammation. Meanwhile, the immunological liver and intestinal injury associated with genetic predisposition.Therefore, the interaction of xenobiotics-gene-environment is becoming increasingly concerned by scientists. We previously have shown that aged i PLA₂β-/- mice have higher incidence of liver cancer and sever intestinal injury compared with aged controls. Since inflammation is one of the essential toxicological factors which contribute to the xenobiotics-induced liver injury, we speculated that the deficiency of i PLA₂β could lead to chronic inflammation that eventually cause age-dependent severe liver and intestinal damage and even hepatocellular carcinoma. We here tested whether deficiency of i PLA₂β could cause sensitization to Con A- and DSS-induced immunological liver and intestinal injury. The related mechanisms for i PLA₂β role in both injuries were systematically discussed in this study, in order to provide a theoretical basis for approaching new precautions and treatments.1. Protective effects of i PLA₂β in Con A-induced immunological liver injuryi PLA₂β knockout and wild-type mice were intravenously injected by 10 mg/kg Con A for 24 h. Histological staining showed that Con A-treated KO mice had larger area of necrosis. We consistently observed an increase in lymphocytes infiltration in mutant liver by immunohistochemistry（IHC） of T cell and B cell markers. We analysed cell proliferation by assessing an S phase maker Ki67 in livers, increased lymphocytes proliferation was observed in mutant liver. IHC staining of α-SMA and Sirius red staining showed that Con A-treated KO mice had greater extend of fibrosis. By IHC staining of cytokeratin 19（CK19）, we consistently observed moderately increased CK19 staining in i PLA₂β-/- livers.These results showed that i PLA₂β have protective roles from Con A-induced liver injury in mice.2. Protective effects of i PLA₂β in Con A-induced intestinal injuryHistological analysis showed that Con A treatment caused severe damage in mutant intestinal epithelial cells associated with exposed lamina propria, shorter villi, disturbed mucus membrane, and mucosal atrophy. We consistently observed an increase in apoptosis in mutant duodenum by IHC and Western blot analysis of Cleaved caspase 3. Con A treatment of mutant mice increased the number of Ki67（+） cells in the duodenal crypts to a greater extent than that of WT mice. Con A treatment of mutant mice caused an increase in jejunal m RNA expression of inflammatory cytokines CD14, TNF-α, IL-6, and SOCS3 and chemokines CCR5, CXCL12 and VCAM1 when compared with Con A-treated WT mice. However, no Con A response difference was observed among WT and mutant intestine for lymphocyte markers. An increased expression of CCL2 protein and m RNA was observed in the jejunum of Con A-treated mutant mice when compared with that in Con A-treated WT mice.We observed AB-PAS blue stains on the mucus membrane layer on the luminal side of intestine. In the duodenum and ileum of Con A-treated mutant mice, there was an increase of blue-stained mucins on the mucus membrane and on the goblet cells. We analyzed two key target genes which related to goblet cell hyperplasia-Klf4 and Ccnd2. We found that Con A treatment of mutant mice increased intestinal Klf4 m RNA expression to a greater extent than that of WT mice. Con A treatment of WT mice induced a marked upregulation of intestinal Ccnd2 m RNA, but failing to so in i PLA₂β-/- mice. The disruption of mucus membrane would allow a leakage of luminal bacteria into intestine and entering into portal vein. Consistently, LPS levels in the portal vein serum obtained from Con A-treated i PLA₂β-/- mice were increased compared with that from Con A-treated WT mice. Hence, our results showed that i PLA₂β have protective roles from Con A-induced intestinal injury in mice.3. Protective effects of iPLA₂β in ConA induced bile acids metablic disorderIn our experimental system, we utilized LC/MS-MS method to determine bile acid concentrations in liver, bile, intestine, feces, and peripheral serum. A net decrease of liver bile acids was obtained with 1303 mg/g.This was consistently observed with a net decrease of bile acids in the bile by 689 mg/m L. A net increase of bile acids by 48 mg/g was observed in the intestine. As for bile acid levels in feces, we observed a net increase of bile acids in peripheral blood from inferior vena cava by 20 mg/m L in Con A treated-i PLA₂b-/- mice. This significant decrease in hepatic and biliary bile acids was associated with suppression of bile acid synthetic gene CYP7A1 which consistent with upregulation of intestinal FXR/FGF15 m RNA expression. Meanwhile, suppression of hepatic bile acids transporter NTCP and BSEP and intestinal bile acids transporter Ostα and ABST was observed in Con A treated-i PLA₂b-/- mice.Hence, our results showed that i PLA₂β have protective roles from Con A-induced alteration in bile acids metabolism in liver and intestine.4. Protective effects of i PLA₂β in DSS induced intestinal injuryAs we have been observed the protective effect of i PLA₂β in the process of liver injury for the small intestine, in order to further confirm the specificity of the immune intestinal injury protection, we continued induction of acute colitis. For an acute colitis induction, 2.5% or 3% DSS was added to drinking water for 7 days, followed by normal tap water for 3 days. The protective role of i PLA₂β in mouse colon was further confirmed by histological analysis. DSS administration of KO mice resulted in an increase in colonic ulceration associated with larger areas of epithelial crypt loss, and inflammatory cell infiltration throughout the mucosa. A decreased number of Ki67（+） and an increased number of Cleaved caspase 3-stained colonocytes were observed in DSS-treated i PLA₂β-/- mice when compared with WT controls. By AB-FR staining, the colon of DSS-treated mutant mice contained less number of mucin-containing goblet cells and the thickness of a continuous inner mucus layer was also drastically decreased.We found a marked increase in infiltrating F4/80（+） macrophages in the lamina propria and F4/80 m RNA expression of DSS-treated KO mice. This was concomitant with increased CD11 b and CD11 c m RNA. In a greater extent in KO compared with WT mice, DSS treatment increased colonic m RNA expression of TNFα, IL-1β, IL-6, MIP-1α, MIP-1β and MCP1. DSS treatment caused a severe damage to the ileum of i PLA₂β-/- mice with a disturbed mucosal architecture, denuded epithelium, exposure of lamina propria, and shorter villi. Consistently, the number of goblet cells was dramatically decreased in the ileum of DSS-treated mutants as seen by AB-FR staining. Without any effects in the WT ileum, DSS treatment of KO mice increased the staining of Cleaved caspase 3 in the ileum found to be located in the crypts. Hence, our results showed that i PLA₂β have protective roles from DSS-induced intestinal injury.