Study On The Treatment Of Tongbi Jiangu Prescription Based On TLR4/NF-κB Signaling Pathway Mechanism Of Action In Treating Knee Osteoarthritis

Objective: Based on TLR4/NF-κB signaling pathway,the effects of Tongbijiangu formula on FLS in KOA model rats and humans were investigated,and the mechanism of action of Tongbijiangu formula in the treatment of KOA was clarified,providing theoretical basis for the clinical use of Tongbijiangu formula in the treatment of KOA.Methods1.There were 50 SD rats,10 were used as blank group,and the remaining 40 were used to construct KOA model by modified Hulth method.After successful modeling,they were randomly divided into model group,Tongbijiangu prescription low-dose,medium-dose and high-dose groups.HE staining was performed on the synovium of knee joint after intervention.The expressions of TNF-α,IL-6 and LEP in serum of each group were detected by ELISA.The expressions of TNF-α and IL-6 protein in the synovium of each group were detected by immunohistochemistry.The expressions of TLR4,My D88,NF-κB,TNF-α,IL-6,LEP and NLRP3 genes in the synovium of each group were determined by RT-PCR.2.Fibroblast-like synovial cells were cultured in vitro,and their morphology was observed under an inverted microscope.Fibroblast-like synovial cells were identified by immunofluorescence staining.Fibroblast-like synovial cells induced by lipopolysaccharide were used to construct KOA models in vitro.The effect of Tongbi Jiangu Formula lyophilized powder on the activity of KOA fibroblast-like synovial cells was determined by CCK-8method.The modeled chondrocytes were divided into model group,Tongbijiangu Fang lowdose,medium-dose and high-dose groups,and the unmodeled chondrocytes were set as blank group.ELISA,immunohistochemistry and RT-PCR were used to detect the expression of indicators in each group from the levels of inflammation,protein and gene,so as to explore the mechanism of Tongbijiangu prescription in the treatment of KOA.Results1.The gross anatomical observation of the knee joint of KOA rats in vivo animal experiments was compared from the aspects of knee swelling and synovial surface luster;In blank group,there was no swelling of knee joint,and the synovium was smooth and bright and reddish.Compared with the blank group,the knee joint of rats in the model group was swollen,the synovial tissue was dark,and the growth was formed.Compared with the model group,knee joint swelling was slight in the high,medium and low drug groups,and the synovium was glossy and transparent.HE staining: The synovial surface of rats in the control group was smooth and flat,and the cells in the inner layer were arranged neatly,with clear layers,and no hyperplasia or congestion.In the model group,the synovial tissue was congested and swollen with obvious inflammatory cell infiltration,and a large number of surface synovial cells migrated to the subintima of the synovial tissue with obvious hyperplasia and hypertrophy.Mucoid hyperplasia suggested that the secretory function of synovial cells was enhanced and inflammatory cell infiltration was obvious.Compared with model group,the synovial surface of rats in Tongbi Jiangu formula groups was relatively smooth,the inner layer cells were arranged in a more orderly manner,the degree of tissue congestion and edema was reduced,no obvious mucoid hyperplasia was observed,and a small amount of inflammatory cells were infiltrated.ELISA results showed that compared with blank group,serum contents of IL-6,TNF-α and LEP in model group were significantly increased,with statistical significance(P<0.05).Compared with model group,the contents of IL-6,TNF-α and LEP in serum of Tongbi Jiangu prescription groups were decreased,with statistical significance(P<0.05).In low,medium and high dose groups,the medium dose group showed the most significant decrease.Immunohistochemical results showed that compared with blank group,IL-6 and TNF-α protein contents in model group were increased,with statistical significance(P<0.05).Compared with model group,IL-6 and TNF-α were decreased in all Tongbi Jiangu prescription groups,with statistical significance(P<0.05).RTPCR results showed that compared with blank group,gene expressions of TLR4,My D88,NF-κBp65,TNF-α,IL-6,NLRP3 and LEP in model group were increased,with statistical significance(P<0.05).Compared with model group,the expressions of TLR4,My D88,NF-κBp65,TNF-α,IL-6,NLRP3 and LEP in Tongbijiangu prescription dose groups were decreased,with statistical significance(P<0.05),and the decrease was most obvious in the medium-dose group.2.In the in vitro cell experiment,cells gradually crawled out from the surrounding tissue after 5 days of primary cell culture.After 10 days of culture,a large number of cells could be observed at the bottom of the flask.When observed under an optical microscope,the cells were fusiform and relatively large.The fibroblast-like synovial cells of the third generation had the same morphology,good growth and clear nuclei.Vimentin protein staining was positive,and the cells were identified as fibroblast-like synoviocytes.Fibroblast-like synoviocytes were induced by lipopolysaccharide to construct KOA models in vitro.The results showed that the fibroblast-like synoviocytes were induced 12,24 and 48 h by LPS in different concentration groups,and the contents of IL-6 and TNF-α were the highest in LPS1ug/m L group for 24 h.Fibroblastoid synoviocytes have the strongest inflammatory response,so they are used as the optimal concentration and timing of intervention to induce inflammatory response models.The results of CCK-8 showed that the optimal intervention time and concentration of Tongbi Jiangu powder on KOA FLS were 48 h and 150μg /m L,respectively.The TLR4/NF-κB signaling pathway and its downstream inflammatory factors were detected by ELISA and RT-PCR from the levels of inflammation,protein and gene,and the results were consistent with animal experiments.Immunohistochemical detection of TNF-α,IL-6,TLR4 and NF-κBp65 showed that compared with blank group,the contents of TNF-α,IL-6,TLR4 and NF-κBp65 protein in model group were increased(P<0.05).Compared with model group,TNF-α,IL-6,TLR4 and NF-κBp65 were decreased in all Tongbijiangu prescription dose groups(P<0.05),and the decrease was most obvious in medium dose group.Conclusion:1.Animal experimental studies have shown that Tongbijiangu Fangfang alleviates KOA by improving inflammatory cell infiltration in joint synovium tissue of KOA rats,alleviating synovium inflammatory response,and reducing the secretion of IL-6,TNF-α and LEP.The mechanism may be related to the regulation of Tongbijiangu Fangfang on TLR4/NF-κB signaling pathway.It can inhibit the expression of TLR4,My D88,NF-κBp65,TNF-α,IL-6,NLRP3 and LEP genes,down-regulate the expression of TNF-α and IL-6 proteins,and reduce the release of downstream inflammatory factors.2.Further cell studies showed that Tongbi Jiangu Fangfang could reduce FLS inflammatory response,regulate TLR4/NF-κB signaling pathway,down-regulate the expression of TLR4,NF-κBp65,TNF-α and IL-6 proteins in the pathway,and inhibit m RNA of this pathway and related factors in the downstream.Finally,the goal is to cure KOA.