Research On The Mechanism Of Blocking Hepatic Fibrosis By Shenzhang Granules Based On PD-1 Regulation Of Kupffer Cells Differentiation

Objective:The study is meant to investigate the distribution of Kupffer cell（KCs）subgroups during the pathogenesis of hepatic fibrosis and whether the differentiations of Kupffer cell subgroups is regulated by programmed cell death protein-1（PD-1）mediated PI3K-Akt signaling pathway.The effects of shenzhang granules on the differentiations of KCs,the expression of PD-1 on the surface of KCs and PI3K-Akt signaling pathway are determined to explore the mechanism of blockage of hepatic fibrosis by shenzhang granules based on PD-1 mediated Kupffer cell differentiations.The previous study revealed the association of activation of hepatic stellate cell with the blockage of hepatic fibrosis blockage by shenzhang granules.Based on it,this study is expected to provide an important clue for exploring potential targets for novel therapeutics of hepatic fibrosis.Methods:（1）40 SD rats were randomly divided into control group（16 rats）and model group（24 rats）.Rats in the model group were intraperitoneal injected with 40%carbon tetrachloride（CCL₄）/vegetable oil 1.0 ml/kg twice a week for 8 weeks.The control group was intraperitoneal injected with vegetable oil at the corresponding time.Four rats were sacrificed in each group after 1W,2W,4W and 8W treatment.（1）Hematoxylin-eosin（HE）staining and Masson staining were performed to analyze the pathological changes of the liver.M1 and M2 cells were identified by immunohistochemical staining with M1 subgroup antibody CD86 and M2subgroup antibody CD163.The levels of IL-1β,IL-2,IL-6,IL-10,TNFαand TGFβ1 in KCs were detected by ELISA.The relationship between the two subgroups and the process of fibrosis was examined at different times.（2）The KCs in different stages of fibrosis were isolated and cultured,and the phagocytic ability of the cells was analyzed by ink phagocytosis test.The proportion of M0,M1 and M2 in the progress of liver fibrosis was detected by flow cytometry.The expression of KCs PD-1 protein during progression of fibrosis was determined by immunofluoresence.（2）Ten SD rats were randomly divided into control group and model group,with 5 rats in each group.Rats in the model group were intraperitoneal injected with 40%CCL₄/vegetable oil 1.0 ml/kg twice a week for 8 weeks.The control group was intraperitoneal injected with an equal volume of vegetable oil.At the end of the 8th week,the rats were sacrificed.Livers were harvested to confirm the success of the liver fibrosis model by HE and Masson staining.（1）The KCs were isolated and cultured.The proportion of KCs M0,M1 and M2 subpopulations was detected by flow cytometry.The expression of PD-1 protein in the two groups were detected by immunofluorescence.（2）The KCs were transfected with PD-1 Si-RNA,and the levels of PI3k,Akt,p-Akt,mTOR and p-mTOR in KCs were detected by Western blot.（3）WT rats（28 rats）and PD-1 knockout（PD-1^-/^-）rats were intraperitoneal injected with CCL₄ to establish liver fibrosis model.The model group was administered with shenzhang granules.These rats were randomly divided into:WT liver fibrosis rat shenzhang granules group（16 rats）,WT liver fibrosis rat control group（12 rats）,PD-1^-/^-liver fibrosis rat shenzhang group（16 rats）,PD-1^-/^-liver fibrosis rat control group（12 rats）.The rats were intragastrically administered for the first time at the first week,and three rats in each group were sacrificed followed 2 hours after the last administration of the second week,the fourth week,and the eighth week.（1）The KCs were isolated and cultured.The proportion of KCs M0,M1 and M2 subpopulations was detected by flow cytometry.（2）The content of IL-1β,IL-2,IL-6,IL-10,TNFα,TGFβ1were determined.（4）Forty healthy male Wistar rats were prepared for drug-containing serum animals,12 healthy SD rats and 12 PD-1^-/^-rats were prepared to establish the model of liver fibrosis.KCs were isolated and cultured after modeling,and were randomly divided into 8 groups:normal control group A,5 times drug concentration group B group,10 times drug concentration group C group,and20 times drug concentration group D group.（1）Flow cytometery was used to detect KCs subpopulations.（2）Real-time PCR was applied to detect the mRNA levels related to PI3K-Akt signaling pathway.（3）Expression and phosphorylation of proteins associated PI3K-Akt signaling pathway were detected by westernblot.（4）KCs cytokine profile was determined by ELISA.Results:（1）HE and Masson staining showed that CCL4 caused obvious liver damage and fibrotic pathological changes.The ink staining test showed that thephagocytosisabilityofKCsgraduallyweakened;the immunohistochemistry and flow typing results demonstrated that M1 type KCs were higher than that of M2 type KCs in the early stage of live fibrosis（1-2W）,and decreased after the 4th peak;M2 type KCs increased slowly in the early stage and exceeded M1 type KCs in the later stage（8W）.ELISA results showed that IL-1,IL-2,IL-6 and TNFαsecreted by M1 showed a trend of increasing first and then decreasing,and reached the highest value at 4W.IL-10 and TGFβ1 secreted by M2 type continued to increase.The immunofluorescence analysis revealed that the expression of KCs PD-1protein in the model group increased rapidly after the second Week,and was significantly higher than the control group after the 4th week（p<0.05）.（2）HE and Masson staining confirmed successful liver fibrosis modeling.The proportion of M1 and M2 in the model group were significantly higher than that in the control group（P<0.05）.Immunofluorescence analysis showed that the expression of KCs PD-1 protein in the model group was significantly higher than that in the control group（p<0.05）.Western blot showed that CCl₄markdly decreased the level of PI3k（P<0.05）.There was no significant difference in the expression of Akt and mTOR between the two groups（P>0.05）,while the levels of p-Akt and p-mTOR were significantly decreased（P>0.05）.The PD-1 knockout significantly increased the level of PI3k（P<0.05）,p-Akt and p-mTOR（P<0.05）,and no significant difference was observed in the level of Akt and mTOR（P>0.05）.（3）Flow cytometry and cytokine profile results showed that M1 and its secreted IL-1,IL-2,IL-6 and TNFαdecreased rapidly,while M2 and its secreted IL-10 and TGFβ1 decreased slowly without the absence of shenzhang granules.M2 and its secreted IL-10 and TGFβ1 decreased,while M1 and its secreted IL-1,IL-2,IL-6 and TNFαincreased after treatment with shenzhang granules.M1 and its secreted IL-1,IL-2,IL-6,TNFαof PD-1^-/^-rats were gradually decreased after modeling,while M2 and its secreted IL-10 and TGFβ1 decreased rapidly.（4）（1）The proportion of M1 subgroup of KCs cells in PD-1 knockout fibrotic model rats was much higher than that in wild type fibrotic model rats,while the proportion of M2 subgroup significantly lower.After treated with drug-containing serum,the proportion of M1 subgroups in KCs was increased remarkably,in which 10-fold concentration group and 20-fold concentration group were significantly higher than 5-fold concentration group.However,there was no significant difference between the 10-fold concentration group and the 20-fold concentration group.Whereas,the proportion of M2 was significantly decreased after the treatment of drug-containing serum.The10-fold concentration group and the 20-fold concentration group were significantly lower than the 5-fold concentration group,but the difference between the 10-fold concentration group and the 20-fold concentration group was not significant（Fig.19-22）.（2）The mRNA expression levels of PI3K,Akt and mTOR,which are the PI3K-Akt signaling pathway-related proteins,in KCs cells of PD-1 knockout fibrotic model rats were significantly higher then that of wild type fibrotic model rats.After treated with drug-containing serum,the mRNA expression level of PD-1 was decreased,in which the 10-fold concentration group and the20-fold concentration group were significantly lower than the 5-fold concentration group.However,there was no significant difference between the10-fold concentration group and the 20-fold concentration group.On the contrary,the mRNA expression level of PI3K,Akt and mTOR proteins was further increased after treated with drug-containing serum,and there was a positive dose-effect relationship.However,there was no significant difference between the 10-fold concentration group and the 20-fold concentration group.（3）The expression of PI3K in KCs cells of PD-1 knockout fibrotic model rats were significantly higher then that of wild type fibrotic model rats,although the expression of Akt and mTOR did not change obviously,but the phosphorylated level of them increased significantly.After treated with drug-containing serum,the expression of PD-1 was decreased,in which the10-fold concentration group and the 20-fold concentration group were significantly lower than the 5-fold concentration group.However,there was no significant difference between the 10-fold concentration group and the20-fold concentration group.On the contrary,the expression of PI3K,p-Akt and p-mTOR proteins was further increased after treated with drug-containing serum,and there was a positive dose-effect relationship at a certain concentration.However,there was no significant difference between the10-fold concentration group and the 20-fold concentration group.（4）The expression levels of cytokines IL-1,IL-2,IL-6 and TNF-alpha,which are secreted by M1 type KCs,were much higher in the liver KCs of PD-1knockout fibrotic model rats then that of wild type fibrotic model rats,while the expression levels of cytokines IL-10 and TGF-beta secreted by M2 type KCs decreased significantly.The expression levels of cytokines IL-1,IL-2,IL-6 and TNF-alpha increased significantly after treatment with drug-containing serum,and there was a positive dose-effect relationship,but there was no significant difference between 10-fold concentration group and20-fold concentration group.On the contrary,the expression levels of IL-10and TGF-beta were significantly decreased,and there was a negative dose-effect relationship,but there was no significant difference between10-fold concentration group and 20-fold concentration group.Furthermore,the results showed that the levels of IL-4 and IFN-gamma in drug-containing serum were higher than those in normal serum.However,the difference of expression level of IL-4 was not significantly,While the expression level of IFN-gamma was significantly increased.Specifically,the expression level of IFN-gamma in 10-fold concentration group and 20-fold concentration group were significantly higher than those in 5-fold concentration group,but there was no significant difference between 10-fold concentration group and 20-fold concentration group.Conclusion:（1）In the early stage of hepatic fibrosis,the proportion of M1-type KCs is dominant,which plays a leading role in cell activation and recruitment;while M2-type KCs increase continually under the activation of M1-type KCs,finally exceeding the M1-type KCs in the late stage of hepatic fibrosis,which directly promotes fibrosis.The decrease of PD-1 expression at early stage of KCs leads to the rapid increase of M1-type KCs,which is involved in the differentiation of KCs subgroup and liver fibrosis.（2）The increase of PD-1 protein expression promotes the differentiation of KCs and induces liver fibrosis.The mechanism is related to the inhibition of PI3K and phosphorylation of Akt and mTOR,which blocks the PI3k/Akt/mTOR signaling pathway.Knockout of PD-1 gene can increase the phosphorylation of Akt and mTOR,activate PI3k/Akt/mTOR signaling pathway,inhibit the differentiation of KCs,and may inhibit the process of liver fibrosis.（3）After modeling,M1-type KCs decreased rapidly in the liver of modeled rats,while M2-type KCs decreased relatively slow.Shenzhang granules treatment inducing the transformation of M2-type KCs to M1-type KCs.There were more M1-type KCs than M2-type KCs in both PD-1^-/-rats and wild type rats,which would decrease gradually after the liver fibrosis model were made.And as a result of knockout of PD1,the M2-type KCs transformated into M1-type KCs,so the M1-type KCs decrease slower in the liver of PD-1^-/-modeled rats than that in wild type modeled rats.Remarkably,PD-1^-/-rats treated with Shenzhang granules could induce M2 to M1 transformation more quickly.（4）The serum containing Shenzhang granules can inhibit the expression of PD-1 and activate PI3K-Akt signaling pathway,and the optimal concentration of serum containing Shenzhang Granule is 10-fold concentration.