Protective Effects Of HO-1/CO On Intestinal Barrier Dysfunction In Cholestatic Liver Injury Through Regulation Of TLR4/NF-?B

Cholestatic liver disease is often caused by liver cells injury,bile duct epithelial cells injury and bile duct obstruction.During cholestasis,the collagen extracellular matrix(Extracellular matrix,ECM)increased while degradation decreased.Excessive ECM deposition in liver perisinusoidal space could induce hepatic fibrosis,even cirrhosis.The progress of liver fibrosis is often accompanied with intestinal flora imbalance,increased intestinal permeability and endotoxemia,which causes the second hit to the liver through the gut-liver axis,forms a vicious circle and aggravates the liver damage.Therefore,the recovery of intestinal barrier is crucial to block the progression of liver fibrosis.When cholestatic liver fibrosis,the reduction of intestinal bile acids often led to the change of the intestinal flora structure and bacterial overgrowth.Some of the pathogenic and pro-inflammatory factors,including the growth of enterobacteriaceae and enterobacteriaceae,increased intestinal permeability.Lipopolysaccharide(LPS)and bacterial translocation caused the inflammatory response,further aggravated intestinal barrier damage.The intestinal epithelial barrier consists of a monolayer of epithelial cells and intercellular junctions between adjacent cells that seal the paracellular space and regulate permeability of the barrier.This barrier is composed of mechanical barrier,immune barrier,biological barrier and chemical barrier,of which,the mechanical barrier is the most important.The intestinal mucosal barrier is a complex and huge defense system,separates harmful luminal substances such as microorganisms,toxins,and antigens from the body and thus plays a critical role in the homeostasis.Our group have continued the study on heme oxygenase 1(HO-1)for a few years.HO-1 is a heat shock protein and the rate-limiting enzyme in the catabolism of heme to yield equimolar amounts of biliverdin(BV),free iron,and carbon monoxide(CO).HO-1 provides protection for cells,tissues,and even whole organs because of its anti-inflammatory and anti-apoptotic functions.Recent studies have found that up-regulation of HO-1 has a protective effect on intestinal barrier.CO is increasingly recognized as a cyto-protective and homeostatic molecule with important signaling capabilities in physiologic and pathophysiologic situations.Transition metal carbonyls,termed CO-releasing molecules(CO-RMs),have been used in biological systems to deliver CO in a controlled manner while keeping carboxyhemoglobin levels stable.CORM-2 is able to transfer CO spontaneously and can exert typical CO-mediated pharmacologic effects.Studies have shown that CORM-2 can protect intestinal epithelial barrier function,inhibit the release of inflammatory cytokines,and promote the recovery of tight junction(TJ)proteins.In this study we observed the protective effect of Co PP(endogenous increase HO-1)and exogenous CORM-2 on LPS induced intestinal barrier injury(including restoring TJ proteins and decreasing the apoptosis of intestinal epithelial cells in Caco-2cell monolayer model.Through construction of lentiviral packaging HO-1overexpression and knockdown stable cell lines,we detected the expression of HO-1,TJ proteins and NF-kappa B,and the results showed that high expression of HO-1 can inhibit the expression of TLR4/NF-kappa B and improve the intestinal TJ protein destruction and reduce the intestinal epithelial cell apoptosis induced by LPS.Finally,the rat model of cholestasis induced liver injury was established by ligation of common bile duct.in vivo experiments further confirmed that Co PP and CORM-2 could protect the intestinal barrier in rats.Based on the above three methods,this study will elustrate the effect of HO-1/CO on intestinal barrier injury during cholestatic liver fibrosis,and provides the experimental basis for the research and development of intestinal barrier drugs.Part ?The protective effects of heme oxygenase-1 and CORM-2 on LPS induced intestinal epithelial barrier dysfunctionObjective: Impaired intestinal barrier function has been implicated in the pathogenesis of many disease,including intestinal inflammatory syndromes and liver disease.The progress of liver fibrosis is often accompanied with intestinal flora imbalance,increased intestinal permeability and endotoxemia,which causes the second hit to the liver through the gut-liver axis,forms a vicious circle and aggravates the liver damage.Therefore,the recovery of intestinal barrier is crucial to block the progression of liver fibrosis.A lot of studies are carried out to explore the underlying mechanisms and evaluating therapeutic strategies for restoring intestinal barrier function.Heme oxygenase-1(HO-1)and its enzymatic by-product carbon monoxide(CO)has been shown to exert a protective effect on intestinal barrier function.In this study we observed the effect of Co PP and CO-releasing molecule-2(CORM-2)on the repairment of intestinal barrier in Caco-2 monolayer.Methods: Firstly,build the Caco-2 cell monolayer and evaluate the integrity of Caco-2 cell monolayer permeability.Caco-2 cells were observed under light microscope,measurement of transepithelial electrical resistance(trans-epithelial electrical,resistance,TEER)and paracellular leakage marker FITC dextran-4000(FD4).To induce barrier dysfunction,Caco-2 monolayers were subjected to lipopolysaccharide(LPS)treatment,in the presence or absence of cobalt protoporphyrin(Co PP)or CORM-2.Transepithelial electrical resistance and paracellular permeability were measured to evaluate barrier function.m RNA and protein levels of HO-1,as well as the TJ proteins occludin,claudin-1 and zonula occludens(ZO-1)were analyzed by real-time PCR and Western blot.Cytokines(IL-6 and TNF-?)levels in the culture medium were detected using ELISA kits.Cell apoptosis was measured by Annexin?/PI staining.The expression of proliferation cell nuclear antigen(PCNA),caspase3 and cleaved caspase3 was determined by Western blot analysis.Results: Caco-2 cells were cultured for 2 weeks,and the cells were observed to form the tight monolayer,TEER maintained at a higher level.LPS could disrupt the intestinal integrity of Caco-2 cell,both the m RNA and protein expression of TJ was significantly decreased.otherwise,the IL-6 and TNF-?production in the culture medium were increased.Co PP could remarkably increased HO-1 expression.upregulation of HO-1(by Co PP)and CORM-2 markedly attenuated the decrease in trans-epithelial electrical resistance and the increase in paracellular permeability in the Caco-2monolayers treated with LPS.Co PP and CORM-2 could reduce LPS-induced cell apoptosis and promote the cell proliferation.Both of them also markedly alleviated the damage caused by LPS manifested by a decrease in the expression of the TJ protein,and inhibited the production of IL-6 and TNF-?.Conclusion: our data suggest that upregulation of HO-1(Co PP)and CORM-2attenuate LPS-induced intestinal epithelial barrier dysfunction by reducing the epithelial cell apoptosis and preventing the damage caused to the TJ protein.Part ?Protective effect and mechanism of HO-1 on intestinal epithelial cellsObjective: To study the effects of HO-1 on intestinal epithelial barrier injury induced by LPS and the possible mechanism of HO-1,clarify the effect of HO-1 on intestinal epithelial cells apoptosis and TJ protein and explore the role of NF-kappa B and its specific inhibitor during the effect.Methods:(1)The cDNA fragment containing the target gene(HO-1)was obtained by RT-PCR and the product of enzyme digestion was connected with FUGW vector and transformed into Trans5 cells.(2)HO-1 sh RNA primer sequence was designed and synthesized,then was connected to PLK0.1 vector after annealing,and the product was transformed into competent cells.(3)The plasmid was extracted after the correct colony was determined,then transfected into 293 T cells,and then the lentivirus carrying HO-1gene and HO-1sh RNA was transfected into Caco-2 cells to form the stable overexpression and knockdown of HO-1/Caco-2 cells.(4)HO-1m RNA and protein level was confirmed by q RT-PCR and Western blot.(5)Establishment of Caco-2 cell monolayer that stable overexpression and knockdown of HO-1.The change of TJ protein ZO-1,claudin-1,occludin was detected after LPS stimulation by RT-PCR,Western blot and immunofluorescence assay.(6)Caco-2 cell apoptosis was detected by flow cytometry and caspase 3 was measured by Western blot.(7)NF-receptor B specific inhibitor JSH-23 was used to treat Caco-2 cells,and the changes of TJ proteins,p65 and p-p65 were observed.Results:(1)the results of sequencing confirmed that HO-1 gene and HO-1shRNA was successfully introduced into FUGW and PLK0.1 vector.RT-q PCR and Western blot confirmed that HO-1m RNA and protein was overexpressed in Caco-2 cells transfected with HO-1 gene.While downregulated in HO-1sh RNA transfected group.(2)Immunofluorescence staining results showed that ZO-1 and claudin-1 protein were expressed in the cell membrane.after LPS stimulation,the fluorescence signal of claudin-1 and ZO-1 was broken,even observed in the cytoplasm.(3)RT-PCR and Western blot results showed that expression of ZO-1 and occludin increased significantly in HO-1 transfected Caco-2 cells compared with the control group,while decreased in sh-HO-1 group.(4)the expression of NF-kappa B was significantly lower in HO-1 high expression group.(5)in normal Caco-2 cells,treated with Co PP(endogenous activation of HO-1)or exogenous supplement of CORM-2 could reduce the expression of TLR4/NF-kappa B induced by LPS stimulation.(6)compared with LPS treated control group,the percentage of early apoptosis in HO-1+LPS group was significantly lower.While it was opposite in LPS treated sh-HO-1 group.(7)In the stable HO-1 overexpressed Caco-2 cells,occludin expression was increased and NF-?B p65 protein was inbibited.In contrast,Caco-2 cells with HO-1 deletion exhibited increased disruption and NF-?B expression compared with the sh-control group.(8)LPS stimulation could induce NF-?B activation,the expression of nuclear NF-?B p65 was increased,while in HO-1 transfected Caco-2 cells,nuclear NF-?B p65 expression decreased.(9)JSH-23 inhibited NF-?B p65 expression,upregulated HO-1 and increased occludin expression.(10)in HO-1 knockdown cell lines,the expression of NF-?B p65 and phospho-NF-?B p65 protein increased significantly compared with HO-1-overexpression group,especially the phospho-NF-?b p65 protein.LPS stimulation can significantly increase the expression of phospho-NF-?B p65 in sh-HO-1group.Conclusion:(1)Recombinant lenti-virus carrying HO-1 gene and HO-1 sh RNA was transfected into Caco-2 cells,the stable Caco-2 cells that overexpression and knock down of HO-1 could be successfully constructed.(2)High expression of HO-1 could significantly improve the expression of ZO-1 and occludin.(3)High expression of HO-1 can reduce the apoptosis of Caco-2 cells induced by LPS.(4)High expression of HO-1 can inhibit the expression of NF-?B p65 activity.(5)JSH-23 can inhibit the expression of NF-?B p65 and up-regulate the expression of occludin.(6)Endogenous significantly increase the expression of phospho-NF-?B p65 in sh-HO-1 group.activation of HO-1 and exogenous CORM-2 supplementation in normal Caco-2 cells can attenuate TLR4/NF-?B expression induced by LPS.(7)LPS stimulation canPart ?Protective effects of HO-1 and CORM-2 on intestinal barrier in rats with cholestatic liver injuryObjective: To observe the changes of intestinal barrier function in rats with cholestatic liver injury induced by bile duct ligation(BDL),and to investigate the protective effect of Co PP and CORM-2 on intestinal barrier.Methods: Sixty SD rats were randomly divided into sham operation group(sham group),model group(BDL group),Co PP treatment group,CORM-2 treatment group and Zn PP treatment group.HE staining was used to observe the pathological changes of liver and ileal mucosa.Serum ALT,AST,AKP,GGT and total bile acid(TBA)levels was measured.ELISA method was used to detect serum endotoxin,D-lactate and DAO activity.The expression of HO-1 and intestinal TJ proteins(ZO-1,claudin-1),apoptosis related proteins(caspase3 and cleaved Caspase3),PCNA,TLR4 and NF-?B p65 protein were detected by Western blot.Immunohistochemical staining was used to observe the distribution of ZO-1,claudin-1.Intestinal TNF-? and IL-6 levels was detected by ELISA.Results:(1)Bile duct ligation can lead to cholestasis,serum TBA level in BDL rats was significantly higher than that in sham group,while the levels of ALT,AST,AKP and GGT were significantly increased.HE staining results showed that BDL rats often accompanied by moderate periportal fibrous hyperplasia and inflammatory cell infiltration.(2)The degree of liver fibrosis in BDL model group was mainly in grade 2,while in Co PP group it was mainly in Grade 1.The degree of liver fibrosis in CORM-2group was significantly lower than that in BDL group.However,there was no significant difference between Zn PP group and BDL group.(3)Pathological examination results showed that the intestinal epithelial cells in BDL group were arranged in disorder,the villi of the small intestine was interrupted,and the mucosa was separated.The morphology of small intestinal mucosa in Co PP group and CORM-2group was relatively complete,and there was no obvious mucosal destruction.According to Chiu's standard on intestinal mucosal injury score,the intestinal mucosal injury in BDL group increased.the injury of intestinal mucosa in Co PP and CORM-2treatment group significantly reduced compared with BDL group.(4)the results of ELISA showed that the level of endotoxin and DAO in BDL group was significantly higher than that in sham group.Serum endotoxin,lactic acid and DAO were significantly lower in CORM-2 treatment group compared with BDL group(P<0.05 or P<0.01).Endotoxin and D-lactate levels in Co PP group was significantly decreased compared with BDL group,while no significant difference was found in DAO levels.(5)Western blot results showed that the expression of ZO-1 and claudin-1 decreased significantly in BDL group than that in the sham.The expression of HO-1 in Co PP group was significantly increased,and the expression of ZO-1 and claudin-1 in Co PP and CORM-2 treatment group were significantly higher than that in BDL group,the difference was statistically significant.(6)Immunohistochemical staining results showed in BDL group,claudin-1 and ZO-1 staining was relatively weak.(7)Western blot showed that the expression of caspase 3 and cleaved caspase3 increased significantly in BDL group,especially the cleaved caspase3.In Co PP and CORM-2group,the expression of cleaved caspase3 was significantly lower than that of BDL group,the difference was statistically significant(P<0.01),there was no significant difference about the expression of caspase 3 and cleaved caspase3 between Zn PP group and BDL group(P>0.05).The expression of PCNA in BDL group was significantly reduced,while in Co PP and CORM-2 treatment group,the expression was significantly higher than that of BDL group,especially in group CORM-2.(8)Western blot results also showed that compared with the sham group,the expression of TLR4 and NF-?B p65 in intestinal tissue of BDL group was significantly increased,Co PP and CORM-2treatment group significantly reduced the TLR4 and NF-?B expression.(9)the level of IL-6 in BDL rats was significantly higher than that in sham group(P<0.05).Compared with BDL group,the expression of IL-6 was significantly lower than that in CORM-2group(P<0.05).Conclusion:(1)BDL rats(cholestatic liver damage)was often accompanied by intestinal barrier dysfunction.(2)HO-1 activation by Co PP and CORM-2 could alleviate liver and intestinal injury,and inhibit the intestinal mucosal epithelial apoptosis.(3)Co PP and CORM-2 could inhibit TLR4/NF-?B expression,promote intestinal epithelial cell proliferation and improve the tight junction,so as to protect the intestinal mucosal barrier function in BDL rats.