Mechanism Research On The Protective Role Of Wutou Decoction Against In Degeneration Of Articular Cartilage In Rat Knee Osteoarthritis

Objective To evaluate the effects of Wu Tou decoction(WTD)on inflammatory signal transduction pathway in KOA articular cartilage degeneration,so as to partly explain its mechanism in the treatment of KOA.Methods 1.Network pharmacology based on molecular docking was used to explore the interactions of Wutou decoction and targets related to an-inflammatory and analgesia actions.2.To induce knee osteoarthritis in Rats used modified Hulth method & specific cold and wet environment,Rats were equally divided into four groups: normal group,model group,WTD group and control group,Based on related intervention,then drawed materials after anesthesia.To observe the pathological changes of articular cartilage by light microscope and scanning electron microscope;The expression of P38 MAPK?JNK?Ras?ERK?i NOS protein were tested by Western Blot;The expression level of IL-1??TNF-? in the synovium were detected by ELISA assay.3.Establishment of chondrocytes system cultured in vitro isolated from knee articular cartilage of 4-week old SD rats;The chondrocytes were identified by toluidine blue and immunohistochemical staining methods;Morphological changes were observed by inverted phase contrast microscope;The effect of viability was dected by MTT assay;the OD value of SOD and NO was dected by enzyme-linked immuno sorbent assay;The expression of P38MAPK?C-myc?C-fos?C-jun?Ras?ERK?i NOS?TNF-a?NF-?B p65?IKK?I?B m RNA and protein were tested by RT-PCR and Western Blot.Results 1.The pharmacodyamic material basis of Wutou decoction mainly have the compound including alkaloids,flavonoids and saponins which can act on targets including inducible p38,JNK,i NOS signal transduction pathway,and have multiple analgesia actions.2.WTD could improve structure and ultrastructure of articular cartilage in KOA;Western Blot revealed that the protein expression levels of P38 MAPK?JNK?Ras?ERK?i NOS in control and WTD groups were significantly downregulated(P<0.05 or P<0.01);ELISA results showed that the expression levels of IL-1??TNF-a in the synovium in control and WTD groups were significantly downregulated(P<0.01).3.The characteristics of the passage 2 chondrocytes were presented: the almost coincident shapes and moderate counts,with clear boundaries and distinct nuclei;amaranthine heterochromatic granules were watched in and around chondrocytes;The cytoplasm of chondrocytes was identified in brownish yellow stained by immunohistochemical staining in positive group;The viability of LPS-induced chondrocytes treated with WTD water extract was enhanced compared to LPS-induced chondrocytes in a time and dose dependent manner(24 h and 150 ?g/m L),as assessed by the MTT assay;Compared with the model group,ELISA suggested that as demonstrated by elevation of the expression of SOD while decline of the expression of NO compared with the model group(P<0.01).WTD water extract(100?150?200 ?g/m L)improved the m RNA and protein expression of P38 MAPK?C-myc?C-fos?C-jun?Ras?ERK?i NOS?TNF-a?NF-?B p65?IKK?I?B,as indicated especially given significant difference from the 150 ?g/m L group(P<0.01).Conclusions 1.Wu Tou decoction(WTD)can inhibits the inflammatory reaction in KOA based on Network pharmacology.2.Wu Tou decoction can improve structure of articular cartilage in the Rats of KOA and relieve the expression of inflammatory factors in articular cartilage,synovium,so as to inhibits articular cartilage degeneration.3.WTD water extract can improve the inflammatory reaction in LPS-induced chondrocytes through regulating on relevant inflammatory signal transduction pathway.