Study On The Treatment And Mechanism Of Pien Tze Huang In Collagen-Induced Arthritis Mice

Objective:This study aimed to explore the therapeutic effect of PZH on collagen-induced arthritis(CIA)mice and its potential mechanism.Methods:1 To establish a collagen-induced arthritis(CIA)model,the DBA/1J mice were induced by type ? collagen of bovine,and the CIA mice were treated by pien tze huang for 4 weeks.The mice were divided into 6 groups: Normal group,Model group,positive control group(MTX,0.30 mg/kg/d),low dose group(PZH-L,0.078 g/kg/d),medium dose group(PZH-M,0.234 g/kg/d)and high dose group(PZH-H,0.702g/kg/d).Body weight,arthritis score,hindpaw width and hindpaw area were recorded twice a week.Serum and ankle joints were collected to store in-80? deep freezer after administration.HE staining was used to detect the pathological conditions of the joints,enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory factors in serum,immunohistochemistry(IHC)was used to detect the expression of inflammatory factors in synovium,and flow cytometry was used to detect the proportion of Th1 and Th17 cells in spleen monocytes.2 After administration,western blot(WB)was used to detect the expression levels of P65,P-P65,I?B?,NOD-like Receptor Protein 3(NLRP3),Apoptosis-associated Speck-like Protein Containing a Card(ASC),Cysteinyl Aspartate-specific Protease-1(Caspase-1)and A20 in the ankle tissues of mice,and IHC was used to detect the expression level of A20 in the synovial tissues of the joints.3 Tartrate Resistant Acid Phosphatase(TRAP)staining was performed to observe the effect of PZH on the number of osteoclasts.The expression of RANKL and OPG in ankle joints of CIA mice were detected by IHC and WB.Paraffin sections of ankle joints in mice were prepared for saffron solid green staining to investigate the pathological conditions of cartilage erosion of CIA mice in each group.Total protein was extracted from the ankle joints of mice,and the expression levels of MMP-3 and MMP-9 were detected by ELISA.Results:1 Efficacy results of PZH on CIA mice: compared with the model group,the measurement results showed that each dose of PZH could effectively improve the mice's clinical symptoms,decrease arthritis score,hindpaw width and hindpaw thickness(P<0.05 or P<0.01 or P<0.001).The histopathological results showed that the mice in model group had a large number of inflammatory cell infiltration in the joint cavity,abnormal synovial tissue proliferation and fibrosis,and severe cartilage and bone damage.The treatment groups could effectively improve the pathological condition and reduce the inflammatory score(P<0.05 or P<0.01)and bone destruction score(P<0.05 or P<0.01).ELISA and IHC results indicated that the expression of IL-1?,IL-6 and IL-17 in the serum and synovium of the model group were significantly higher than those in the normal group.The PZH treatment groups could effectively reduce the expression levels of IL-1?,IL-6 and IL-17 in the serum and joint synovium.(P<0.05 or P<0.01 or P<0.001).Flow cytometry results showed that the proportion of Th1 and Th17 cells in spleen monocytes in model group was significantly higher than that in normal group,and the proportion of Th1 and Th17 in CIA mice could be reduced after treatment of PZH(P<0.05 or P<0.01 or P<0.001).2 Results of anti-inflammatory mechanisms of PZH: WB and IHC results showed that the expression of P-P65,NLRP3,ASC and caspase-1 were obviously higher than that of normal group,the I?B? and A20 expression significantly lower than the normal group and PZH could effectively reduce the expression of P-P65,NLRP3,ASC,caspase-1 in ankle joints,raise the the expression level of I?B? and A20,and the data were significantly different(P < 0.05 or P < 0.01 or P < 0.001),while there was no significant different of PZH to adjust the P65.3 Effect of PZH on bone damage: TRAP staining displayed that the number of osteoclasts in the model group increased significantly and the number in the PZH groups could be reduced(P<0.05 or P<0.01).WB and IHC results revealed that the expression of RANKL in the ankle joints of the model group was significantly increased,while the expression of OPG was significantly decreased compared with the normal group.The PZH group could effectively inhibit the expression of RANKL,and adjust the level of OPG,compared with the model group(P<0.05 or P<0.01 or P<0.001).The results of saffron solid green staining showed that in the model group, the cartilage in the ankle joints was multiple destruction,and the chondrocytes were almost dead,and the cartilage matrix was degraded seriously.The PZH administration groups could significantly relief the cartilage damage.ELISA results indicated that the expression of MMP-3 and MMP-9 in the ankle joints of the model group were significantly higher than that of the normal group,and the expressions of these matrix mtalloproteinases in each PZH groups were effectively reduced(P<0.05 or P<0.01).Conclusion:1 PZH could effectively treat CIA mice by reducing inflammation.2 The mechanism of PZH was closely related to the inhibition of NF-?B signaling pathway and the activity of NLRP3 inflammasome.3 PZH had a significant therapeutic effect on bone damage and cartilage erosion in CIA mice,and the mechanism was closely related to the regulation of expression of RANKL/OPG and matrix metalloproteittases.