Hepatic Stellate Cell Autophagy Induced By Clonorchis Sinensis Is Involved In The Study Of Hepatic Fibrosis

Objectives To clarify whether Clonorchis sinensis infection can induce hepatic stellate cell autophagy,and further elucidate the role of the change of autophagy level in the occurrence of liver fibrosis caused by Clonorchis sinensis infection.Methods1.Study on the relationship between autophagy and hepatic fibrosis induced by Clonorchis sinensis infectionTo construct a model of Clonorchis sinensis infection,SD rats were divided into experimental group and control group,with 10 in each group.Rats in the experimental group were infected with 100 metacercariae by oral gavage,and rats in the control group were given the same amount of normal saline by gavage.Rats were killed 24 weeks after infection,the histological changes and fibroplasia of the liver tissue were observed by paraffin embedded sections,HE staining and Masson staining.The liver tissue was graded according to the fibrosis stage of Metavir.Total protein was extracted from liver tissue by shearing,grinding,and shock crushing.Changes of LC3,P62,and α-SMA of autophagy and fibrosisrelated proteins in liver tissue were detected by Western blot.2.Effects of Clonorchis sinensis excretory/secretory proteins(ESP)on hepatic stellate cell autophagyAdult of Clonorchis Sinensis were obtained by oral gavage in rabbits.PBS sterile culture,Culture medium for 6 h and 12 h was collected;Complete and active adult worms were collected and centrifuged with automatic tissue crushing apparatus after crushing.Centrifuge,take the supernatant,filter and sterilize it,and then separated into adult proteins of Clonorchis Sinensis(Cs TP).Lx-2 cells were transfected with double fluorescent label e GFP-m RFP-LC3 plasmid to observe autophagy.The transfected cells were divided into the blank group,the transfected control group,6 h Cs ESP group,12 h Cs ESP group,the Cs TP group and the positive control group.After the routine culture for 6 h,the cells were photographed and counted by fluorescence microscope for statistical analysis.SDS-page analysis of protein components of 6 h Cs ESP,12 h Cs ESP and Cs TP.To explore the reasons for the difference of autophagy induced in each group.3.Study on the role of autophagy induced by Clonorchis sinensis ESP in hepatic fibrosisRoutine culture of lx-2 cells in vitro,ESP according to 25 μg/ml,50 μg/ml,100 μg/ml concentration gradient in culture medium,control group to join PBS,6 h,12 h,24 h,36 h and 48 h samples were collected,Western blot detection LC3Ⅱ/Ⅰ protein ratio changes determine Lx-2 cell autophagy induced by Cs ESP and point in time;The combination of Cs ESP and chloroquine stimulated LX-2 cells,demonstrating from another perspective that Cs ESP activated LX-2 cell autophagy.Sample total protein extraction LX-2 cells,Western blot detection LC3Ⅱ/Ⅰ protein ratio changes observed between groups autophagy;To observe the effect of Cs ESP on the expression of liver fibrosis related genes in LX-2 cells,the experimental group was supplemented with 10% FBS medium containing 100μg/ml Cs ESP,and the control group was supplemented with 10% FBS medium.Total RNA samples were collected at 6 h,24 h and 48 h,The q RT-PCR detection of liver fibrosis related genes Collagen-Ⅰ,Collagen-Ⅲ,α-SMA expression changes;Chloroquine was used to intervene in the autophagy process to verify that Cs ESP was involved in the regulation of liver fibrosis related genes through autophagy,The experiment was divided into Cs ESP treatment group,chloroquine+ Cs ESP treatment group and blank group.Lx-2 cells were collected at 6,24,and48 h,and total RNA was extracted.q RT-PCR detection of liver fibrosis related genes Collagen-Ⅰ,Collagen-Ⅲ,α-SMA expression changes,the results for statistical analysis.Results1.Clonorchis sinensis was detected in all SD rats infected with SD,and the rat model of infection was successfully constructed.The pathological results of the infected group indicated fibrosis of liver tissue.According to the Metavir scoring system,7 rats in the control group were at the F0 stage and only 3 were at the F1 stage.In the infected group,4 rats were in stage F1 and 6 were in stage F2.Western blot results showed that 6 of the 10 rats in the infection group had increased autophagy level,and the expression level of fibros-related proteins also increased,indicating a correlation between the two.There may be a relationship between autophagy and hepatic fibrosis after Clonorchis sinensis infection.2.In the fluorescent transfection experiment,compared with the 6 h Cs ESP group,12 h Cs ESP group,and the Cs TP group,the red and yellow fluorescence Numbers decreased successively,and the activation of autophagy decreased successively,but they were all higher than the control group(P<0.01).Protein electrophoresis results showed that the main protein bands of 6h Cs ESP and 12 h Cs ESP were around 26 KDa,and the Cs TP were distributed in a waterfall pattern.The main protein bands were around 70 KDa and 26 KDa.The protein contents of6 h Cs ESP,12 h Cs ESP and Cs TP decreased successively at about 26 KDa,which was consistent with the change of autophagy activation ability in each group,and the two may be correlated.Clonorchis sinensis ESP has a stronger ability to activate autophagy.3.The results of Western blot showed that the autophagy level of LX-2 cells treated by Cs ESP increased at 6 h and 48 h,and autophagy was not activated at other time points.The LC3-Ⅱ/LC3-Ⅰ ratio of CQ + Cs ESP group increased compared with CQ group,and Cs ESP could induce autophagy.q RT-PCR detection results show that the Cs ESP stimulus LX-2 cells for 48 h,Collagen typeⅠ and Collagen type Ⅲ m RNA expression increases.Cs ESP combined with chloroquine stimulated LX-2 cells,and Cs ESP restored the decreased expression of genes associated with liver fibrosis caused by chloroquine inhibition of autophagy.ConclusionIt is clear that Clonorchis sinensis excretory/secretory proteins(ESP)can activate hepatic stellate cell autophagy,and induced autophagy can promote the expression of liver fibrosis related genes,indicating that autophagy plays a certain role in the occurrence of liver fibrosis caused by Clonorchis sinensis infection.