Effect Of CHI3L1 On Hepatocyte Apoptosis In Brain Death And Its Mechanism

Part 1 Expression and significance of CHI3L1 in liver under brain deathObjectiveTo investigate the expression of chitinase-3-like protein 1(CHI3L1)and protease-activated receptor 2(PAR2)in liver under brain death.Methods1.Liver tissue samples from brain-dead donor liver before cold perfusion of clinical liver transplantation and normal liver tissue of control group were collected(n=8).RNA-seq was used to analyze whether CHI3L1 is a differentially expressed gene in brain-dead donor liver(n=3),and qRT-PCR was used to detect the changes of CHI3L1 gene expression(n=8).2.Livers of rats with brain death for 6 hours were studied.Twelve male rats were randomly divided into Brain death group(BD)and sham-operated group(Sham).The donor liver stress model of rat brain death was established.The gene levels and protein expression changes of CHI3L1,PAR2 and iNOS in the donor liver tissues of rat brain death were detected by qRT-PCR,Western blot and immunohistochemical staining,and the main cell sources of CHI3L1 in the donor liver tissues of rat brain death were detected by immunofluorescence double-labeled staining.3.The polarized macrophage model of THP-1 monocyte line and the hypoxia and reoxygenation model of L0-2 liver cell line were established.The stress process of macrophages and hepatocytes in the pathological state of liver ischemia and reperfusion of brain-dead donors was simulated by above two models,respectively.liver ischemia and reperfusion of brain-dead donors,respectively.The phenotypes of M1 and M2 macrophages were identified by flow cytometry antibody labeling.The gene levels and protein expression changes of macrophages in different polarization states were detected by Western blot and qRT-PCR respectively,and protein expression changes of hepatocytes after hypoxia and reoxygenation were detected by Western blot.Results1.The expression of CHI3L1 was significantly up-regulated in clinical brain death donor liver tissue samples:RNA-seq differential expression gene analysis showed that the expression of CHI3L1mRNA was up-regulated 7.74 folds in human brain death donor liver(n=3,P<0.05).Further qRT-PCR detection showed that mRNA of CHI3L1 was significantly up-regulated in human brain dead donor liver,increasing by 9.09 folds(n=8,P<0.05).2.High expression of CHI3L1 in brain dead donor liver macrophages:Compared with the sham-operated group,the mRNA expression level of CHI3L1 in the liver tissue of brain-dead rats was increased by 3.12 folds(n=6,P<0.05).Immunohistochemical staining of liver showed that the expression of CHI3L1 was also increased.Western blot analysis showed that the relative expression of CHI3L1 protein increased by 2.17 folds(n=6,P<0.05).The expression of CHI3L1 was localized in macrophages in the liver by immunoco-fluorescence double-label staining of rat brain dead liver tissue.The expression of iNOS mRNA,the polarization marker of M1 macrophages,was significantly increased in the liver tissue of rats after brain death 6 hours,suggesting that the polarization of M1 macrophages appeared in the liver in the state of brain death.3.CHI3L1 was highly expressed in M1-polarized macrophages:THP-1 was successfully polarized into Ml-type(CD14+CD80,CD14+CD86)and M2-type(CD14+CD163)macrophages by flow cytometry antibody labeling.The qRT-PCR results showed that the mRNA expression of CHI3L1 was the highest in M1 macrophages.Western blot analysis showed that the expression of CHI3L1 protein in LO-2 liver cells was significantly lower than that in macrophages with different polarization states,and the expression of CHI3L1 protein was the highest in M1 type macrophages(P<0.05).4.PAR2 expression was up-regulated in brain-dead donor liver tissue of rats:Compared with sham operation group,PAR2 mRNA expression in liver of brain dead rats was increased by 4.9 folds(n=6,P<0.05).Immunohistochemical staining of liver showed that PAR2 expression was significantly increased at 6h after brain death.Western blot results showed that the expression of PAR2 protein with PAR2 mRNA were analogously increased(n=6,P<0.05).Conclusions1.For the first time,this part of the experiment found that the expression of CHI3L1 was increased in the liver of brain-dead donors at the clinical level,in vivo and in vitro,and the high expression of CHI3L1 protein in macrophages dominated by MI type may be involved in the liver injury in brain death.2.Combined with qRT-PCR analysis,immunohistochemical staining and Western blot PAR2 expression was significantly increased in brain dead liver tissue of rats,which provided experimental basis for further study on the biological effects of PAR2-mediated CHI3LI on liver cells.Part 2 Effect and mechanism of CHI3L1 on hepatocyte apoptosisObjectiveTo investigate the biological effects of CHI3L1 on hepatocyte apoptosis and its signaling pathway,and to find therapeutic targets for the protection of hepatocyte apoptosis in brain death state.Methods1.After Hypoxia/reoxygenation(H/R)stress model was established,the biological effects of recombinant CHI3L1(rCHI3L1)on hepatocytes were observed.Hepatocytes in the positive control group were treated with protease-activated receptors 2-activating polypeptide(PAR2-AP).For si-PAR2(Small interfering RNA targeted to PAR2)hepatocyte group were transfected with siRNA cell to knockdown PAR2 receptor,and the hepatocytes in the S-NC(Small RNA targeted to negative control)group served as siRNA negative control.CCK-8 detect Control(normoxia or H/R),rCHI3L1(100ng/mL),PAR2-AP(500μpM),rCHI3L1+Si-NC and rCHI3L1+Si-PAR2 groups.The survival rates of liver cells were analyzed in each group under normoxia culture and hypoxia reoxygenation stress,respectively.2.LO-2 hepatocytes from Control(simple normoxia or H/R hepatocytes),rCHI3L1(l00ng/mL)and PAR2-AP(500uM)groups were collected under normoxia and H/R culture conditions,and qRT-PCR and Western blot were used to detect the gene expression levels and protein expression of PAR2 and Caspase3 in liver cells treated with different measures.3.Control(simple normoxia or H/R hepatocytes),Si-NC and Si-PAR2 were analyzed by Western blot.The changes of PAR2 and Caspase3 protein expression in liver cells of each group under normoxia and H/R conditions were detected respectively,in order to clarify the effect of PAR2 receptor itself on Caspase3 and its lysate protein expression.4.To investigate the activation of PAR2 receptor and the effect of CHI3L1 on the expression of apoptotic proteins.Western blot was used to detect the expression of PAR2,Caspase3 and Cleaved-caspase3 protein in Control(normoxia or H/R),rCHI3L1+Si-NC and rCHI3L1+Si-PAR2 groups under normoxia and H/R stress,respectively.5.LO-2 hepatocytes were collected from Control(simple H/R hepatocytes),H/R+rCHI3L1(100ng/mL)and H/R+PAR2-AP(500uM)groups under H/R stress condition,and Western blot was used to screen the signaling pathway proteins such as MAPK,AKT,STAT3 and NF-κB/p65 that might be associated with CHI3L1 inducing cell death.Then,CHI3L1(10ng/mL)and PAR2-AP(500uM)were used to act on PAR2,respectively.SiRNA knockdown of PAR2,20μM SP600125 inhibition of JNK,and 20μM U0126 inhibition of ERK were used to study the signaling molecular pathway proteins.AnnexinV/PI flow cytometry was used to detect the changes in apoptosis rate of liver cells.Results1.SiRNA knockdown of PAR2 receptor in hepatocytes alleviates the damage of CHI3L1 to hepatocytes:CCK-8 detection revealed that the effect of rCHI3L1,PAR2-AP,Si-PAR2 on the survival rate of LO-2 cells had a trend of change in normal oxygen culture conditions,but the difference was not statistically significant.However,after LO-2 cells were subjected to hypoxia/reoxygenation,100ng/mL rCHI3L1 significantly reduced the survival rate of LO-2 hepatocytes,and the inhibitory effect of 500μM PAR2-AP positive control group and Si-NC blank control group on survival rate of hepatocytes showed consistent decrease(P<0.05).When siRNA knocked down the PAR2 receptor in LO-2 hepatocytes,it was found that the inhibitory effect of rCHI3L1 on the survival of H/R hepatocytes was completely restored by Si-PAR2.2.CHI3L1 consistently up-regulated the expression levels of PAR2 mRNA and Caspase3 mRNA in liver cells:qRT-PCR results showed that LO-2 cells were both in the normal oxygen culture group and in the hypoxia/reoxygenation stress group,rCHI3L1 significantly up-regulated the expression of PAR2 and Caspase3 genes.The effect of 100ng/mL rCHI3Ll on the expression of PAR2 receptor gene in hypoxia stress liver cells was even greater than that of 500μM PAR2-AP positive control,and the up-regulated levels of PAR2 mRNA and Caspase3 mRNA showed consistency changes(P<0.05).3.Activation of PAR2 receptor in liver cells is involved in the expression of Caspase3 protein induced by CHI3L1:Western blot analysis showed that rCHI3L1 has a positive inducing effect on PAR2 and Caspase3 in both LO-2 cells under normal oxygen culture and hypoxia/reoxygenation stress culture conditions.The expression of PAR2.Caspase3 and Cleaved-caspase3 protein was significantly up-regulated by induction(P<0.05).Without the addition of rCHI3L1,Si-PAR2 had a significant knockdown effect on the expression of PAR2 receptor in both normoxic cultured liver cells and hypoxic stress liver cells(P<0.05).However,the corresponding Caspase3 and Cleaved-caspase3 proteins were not reduced in the Control,Si-NC,and Si-PAR2 groups in both normoxic and hypoxic stress conditions.When rCHI3L1 was added,the PAR2 receptor of normoxic cultured liver cells and hypoxic-stressed liver cells was knocked down,and the results showed that Caspase3 protein and Cleaved-caspase3 protein were consistently down-regulated in corresponding Si-PAR2 groups(P<0.05).4.CHI3L1 activates MAPK/JNK and ERK signals in liver cells:Western blot analysis showed that rCHI3Ll did not significantly activate AKT,STAT3 and NF-κB/p65 signaling proteins in H/R hepatocytes,as did PAR2-AP positive control,but rCHI3L1 simultaneously activated p-JNK and p-ERK signaling proteins in MAPK signaling pathway(P<0.05).while rCHI3L1 had no significant effect on P38 and p-P38.However,the positive control PAR2-AP only activated p-JNK,but did not activate p-ERK.5.CHI3L1-PAR2 signaling is involved in the regulation of liver cell apoptosis by activating MAPK/JNK:Western blot detection was performed on LO-2 liver cells under hypoxia and reoxygenation stress state supplemented with rCHI3L1 and PAR2-AP.In Si-PAR2 group,p-JNK protein level was significantly decreased(P<0.05),but p-ERK protein level was not significantly changed.Annexin V/PI flow cytometry results showed that the change of hepatocyte apoptosis rate in the Control,Si-NC and Si-PAR2 groups was not consistent with the change of p-ERK protein level,but was consistent with the change of p-JNK protein level.6.JNK phosphorylation is involved in regulation of CHI3L1 induced hepatocyte apoptosis:SP600125 was applied to inhibit JNK phosphorylation level on the basis of stimulating hypoxia stress hepatocytes with rCHI3L1 and PAR2-AP positive control respectively,and the results showed that p-JNK protein expression in LO-2 hepatocytes was effectively inhibited under the H/R stress state.Annexin V/PI flow cytometry results showed that the apoptosis rate of liver cells decreased synchronically after SP600125 effectively inhibited p-JNK(P<0.05).The results were consistent with the PAR2-AP positive control.7.The activation of ERK phosphorylation by CHI3L1 has nothing to do with the molecular mechanism of inducing apoptosis of hypoxic stress hepatocytes:On the basis of stimulating hypoxic stress hepatocytes by rCHI3L1,U0126 was applied to inhibit ERK phosphorylation level,and the results showed that p-ERK protein expression in LO-2 hepatocytes was effectively inhibited under hypoxic stress state(P<0.05).Annexin V/PI flow cytometry results showed that after U0126 effectively inhibited p-ERK,but the apoptosis rate of liver cells did not decrease synchronically.Conclusions1.This part of the experiment found for the first time that CHI3L1 has a biological function of promoting apoptosis in hypoxic stress hepatocytes through in vitro studies,and this biological effect is mediated by the activation of PAR2 receptor on the surface of hepatocytes.2.Combined with CCK-8,qRT-PCR,Western blot and Annexin V/PI cell apoptosis detection,the complete CHI3L1-PAR2-MAPK/JNK-Caspase3 hepatocyte apoptosis signaling molecular pathway was revealed for the first time.Part 3 Targeting inhibition of CHI3L1/PAR2 to protect effect of hepatocyte apoptosis under brain deathObjectiveTo verify the regulation of CHI3L1 target on hepatocyte apoptosis signaling pathway in vivo,providing a new and effective prevention and treatment strategy for the occurrence of hepatocyte apoptosis in the state of brain death.MethodsThe rats with brain death(BD)for 6h and chitin intervention for BD 6h were randomly divided into four groups.Twenty-four male rats were randomly divided into Brain death(BD)group,Sham-operated group(Sham)group,and Chitin intervention Brain death group for 6h(Chitin+Brain death,CB)and Chitin intervention Sham-operated(Chitin+Sham,CS)group.With 30 min pretreatment before brain death induced animal,CB group rats femoral venous indwelling needle injection preparation chitin particles(10mg:1mL)PBS suspension liquid.After 30 minutes,begining to slowly intracranial pressure induced brain death,The CS group rats with CB intravenous injection in rats the same dose of chitin particle and the same time of action.Liver samples were collected at the end of the experiment when brain death lasts for 6h.Immunohistochemical staining was used to detect the protein expression changes of CHI3L1 and PAR2 in liver tissues of rats under brain death and after chitin intervention.Western blot was used to detect protein expression levels of CHI3L1,PAR2,JNK,p-JNK,Caspase3 and Cleaved-caspase3 apoptotic signaling molecules in the liver tissue under brain death.The changes of liver cell apoptosis in rats with brain death and after chitin intervention were detected by TUNEL fluorescence double staining.Results1.Chitin inhibited the expression of CHI3L1 protein in the liver of brain-dead rats:Liver immunohistochemical staining(IHC)and Western blot results showed that compared with Sham group and CS group,the expression of CHI3L1 protein in the liver of BD group was significantly increased,and the expression level of CHI3L1 protein in the liver of CB group was significantly decreased after intravenous injection of chitin(n=6,P<0.05).2.Chitin inhibited the expression of PAR2 protein in the liver of brain-dead rats:Liver immunohistochemical staining(IHC)and Western blot results showed that compared with Sham group and CS group,the expression of PAR2 protein in the liver of rats in the BD group was significantly increased,and the expression of PAR2 protein in the liver of rats in the CB group after intravenous injection of chitin was significantly decreased(n=6,P<0.05).3.Chitin reduced liver cell apoptosis in brain-dead rats by inhibiting the target of CHI3L1/PAR2:Western blot results showed that the protein expressions of p-JNK,Caspase3 and Cleaved-caspase3 in the liver tissues of the BD group were increased.Compared with the BD group,the protein expression levels of p-JNK,Caspase3 and Cleaved-caspase3 in liver tissues of the CB group were significantly decreased after intravenous administration of chitin in brain-dead rats(n=6,P<0.05).The results of TUNEL fluorescence double staining showed that the apoptosis rate of liver cells in each group was correlated with the CHI3L1-PAR2-MAPK/JNK-Caspase3 molecular pathway proteins.The protein expression of CHI3L1-PAR2-MAPK/JNK-Caspase3 molecular pathway was consistent with the apoptosis rate of liver cells in each group,and the apoptosis rate of liver cells was significantly decreased after intravenous injection of chitin in brain-dead rats(n=6,P<0.05).Conclusions1.This part of the experiment was proved by in vivo study that CHI3L1-PAR2-MAPK/JNK-Caspase3 hepatocyte apoptosis signaling molecular pathway is involved in the occurrence of hepatocyte apoptosis in brain death state.2.Chitin targeting inhibition of CHI3L1/PAR2 signal can alleviate the occurrence of hepatocyte apoptosis in the state of brain death.