| Aim:To establish an autoimmune hepatitis(AIH)mouse model by tail vein injection of Con A,to clarify the therapeutic effect of Pien Tze Huang(PZH)on AIH,and to explore its possible mechanism from the perspective of regulating macrophage polarization.Methods:1.Animal model replication and administration methods:60 SPF male C57BL/6J mice were randomly divided into 6 groups,namely normal group,model group,low,medium and high dose group(PTH-L,PTH-M,PTH-H)and dexamethasone(DXM)group,with 10 mice in each group.After 3 days of adaptive feeding,drugs were administered at fixed intervals daily for a total of 10 days of prophylactic administration.The normal group and the model group were treated with the same volume of 0.9%sodium chloride solution by gavage,the Pien Tze Huang low,medium and high dose groups were treated with Pien Tze Huang 117 mg/kg,234 mg/kg and 468 mg/kg by gavage,respectively,and the dexamethasone group was treated with dexamethasone 3 mg/kg by intraperitoneal injection.Three hours after the last administration,the AIH model was established by tail vein injection of Concanavalin A(Con A).Except for the normal group,which was injected with 0.2 ml 0.9%sodium chloride solution,the other groups were injected with Con A 15 mg/kg through the tail vein once,and fasted without drinking.After 8 hours,the mice were sacrificed and peripheral blood,liver and spleen tissues were collected.2.Efficacy evaluation of Pien Tze Huang on concanavin A-induced AIH mice:the changes of body weight,hair color,degree of activity and mental state of mice in each group were observed and recorded at the same time every day.The mice were anesthetized and euthanized,and the spleen and liver tissues were harvested and weighed.Spleen weight,liver weight,spleen weight index and liver weight index were calculated.The liver pathological sections were prepared,and the liver pathological conditions were observed under the light microscope.The double-blind method was used to score the pathological damage of the liver.The peripheral blood serum of mice was prepared for liver function test,and the indicators were ALT,AST,DBIL,IBIL,TBIL,ALP,ALB,GOLB,CHOL,GGT,LDL-C,HDL-C,TP and TG.In addition,the levels of IL-4,IL-10,TGF-β,IL-6,IL-12,IL-17 and IFN-γ cytokines in mouse liver tissues were measured by Elisa.3.Regulatory effect of Pien Tze Huang on M1/M2 macrophages in AIH mice:The levels of M1 macrophages(CD11b+F4/80+iNOS+、CD11b+F4/80+TIM-1+、CD11b+F4/80+TLR4+和 CD11b+F4/80+MHC-II+)and M2 macrophages(CD11b+F4/80+CD163+、CD11b+F4/80+CD206+)in the liver tissue of mice were detected by flow cytometry to explore the immune mechanism of Pien Tze Huang in the treatment of AIH.4.Regulation of Pien Tze Huang on mTOR signaling pathway in AIH mice:Western blot method was used to detect the expression of mTOR signaling pathway-related proteins to explore the regulation effect of Pien Tze Huang on mTOR signaling pathway in AIH mice.Results:1.Pharmacodynamic evaluation of Pien Tze Huang on concanavin A-induced AIH mice:Compared with the normal group,the mice in the model group showed significant weight loss,significant listlessness,decreased appetite,and damaged hair gloss in the later stage.The weight of spleen and liver,liver weight index and spleen weight index also increased significantly(p<0.01).Compared with the model group,the symptoms of the treatment group were alleviated,and the spleen weight,liver weight,liver weight index and spleen weight index were significantly decreased(p<0.01 or p<0.05).The results suggest that Pien Tze Huang can effectively improve the symptoms of experimental AIH mice.Under the microscope,the pathological sections of the liver showed that the liver structure of the normal group was clear,the hepatocytes were intact,and the hepatic cords were arranged neatly without obvious abnormalities.Compared with the normal group,the liver lobular structure of the model group was seriously damaged,the arrangement of hepatic cords was disordered,a large number of inflammatory cell infiltration,partial necrosis,and rosette like changes were observed.The pathological injury score of the model group was also significantly increased(p<0.01).However,the low-middle-high-dose Pien Tze Huang group and dexamethasone group had different degrees of repair of liver structure,reduced inflammatory infiltration and necrosis of liver cells,and significantly decreased pathological injury scores(p<0.01 or p<0.05).Liver function tests showed that compared with the normal group,the serum levels of ALT,AST,DBIL,IBIL,TBIL,ALP,LDL-C and TG in the model group were significantly increased(p<0.01).Compared with the model group,the levels of the above liver function indexes in each treatment group were significantly decreased(p<0.01 or p<0.05).At the same time,compared with the normal control group,the levels of ALB,GLOB,CHOL,GGT,HDL-C and TP in the serum of the model group were significantly decreased(p<0.01 or p<0.05),while the corresponding liver function indexes in each treatment group were also increased to varying degrees(p<0.01 or p<0.05).Elisa detection of anti-inflammatory cytokines and pro-inflammatory cytokines in the liver tissue showed that the expression levels of IL-4,IL-10 and TGF-β in the model group were significantly lower than those in the normal group(p<0.01).However,the expression of IL-6,IL-12,IL-17 and IFN-γ was significantly increased(p<0.01).However,the expression levels of IL-4,IL-10 and TGF-β in each treatment group were significantly increased(p<0.01),and the expression levels of IL-6,IL-12,IL-17 and IFN-γ were significantly decreased(p<0.01 or p<0.05)after the treatment with low,medium and high doses of Pien Tze Huang and dexamethasone.2.Regulatory effects of Pien Tze Huang on macrophages in AIH mice:Compared with the normal group,the number of M1 macrophages CD11b+F4/80+iNOS+、CD11b+F4/80+TIM-1+、CD11b+F4/80+TLR4+and CD11b+F4/80+MHC-Ⅱ+ cells in the model group was significantly increased(p<0.01).However,the number of CD11b+F4/80+CD163+、CD11b+F4/80+CD206+M2 macrophages was significantly decreased(p<0.01).At the same time,mice in each treatment group after treatment with Pien Tze Huang medium dose and dexamethasone,The number of M1 macrophages CD11b+F4/80+iNOS+、CD11b+F4/80+TIM-1+、CD11b+F4/80+TLR4+and CD11b+F4/80+MHC-Ⅱ+ cells was significantly decreased(p<0.01 or p<0.05).However,the number of CD11b+F4/80+CD163+、CD11b+F4/80+CD206+M2 macrophages was significantly increased(p<0.01).The results suggest that Pien Tze Huang can effectively regulate the balance of M1/M2 macrophages in autoimmune hepatitis mice.3.The effect of Pien Tze Huang on mTOR signaling pathway in AIH mice:Compared with the normal group,the expression level of P53 in liver tissue of the model group was significantly decreased(p<0.01),while the protein levels of ERK1/2,Akt,p-Akt and PI3K were significantly increased(p<0.01).In the Pien Tze Huang and dexamethasone treatment groups,the expression level of P53 was significantly increased(p<0.01),while the protein levels of ERK1/2,Akt,p-Akt and PI3K were significantly decreased(p<0.01 or p<0.05).It is suggested that Pien can effectively inhibit the activation of mTOR signaling pathway in AIH mice.Concussion:1.Pien Tze Huang can effectively alleviate the symptoms of AIH mice induced by Con A.2.The middle-dose Pien Tze Huang is effective in the treatment of AIH.3.Pien Tze Huang can effectively regulate the balance of M1 and M2 macrophages in peripheral blood of AIH model mice,which may be related to the inhibition of mTOR signaling pathway activation. |
PDF Full Text Request