Study On The Mechanism Of Yougui Pills In Mediating Cartilage Proliferation And Differentiation In Knee Osteoarthritis Based On MEK/ERK Signaling Pathwa

Objectives: Network pharmacology was used to explore the signal pathway of Yougui pills in the intervention of knee osteoarthritis.To explore the intervention of Yougui Wan on knee osteoarthritis model rats,observe the effect of Yougui Wan on the proliferation and differentiation of knee arthritis cartilage and MEK/ERK signaling pathway in synovium cells of rats,explore the expression of this pathway and the mechanism of prevention and treatment of knee osteoarthritis,which is for the clinical prevention and treatment of knee osteoarthritis.And the follow-up study provides theoretical support.Material and methods:1 All active ingredients and target proteins of Yougui Wan were screened using TCM Systematic Pharmacology Database and Analysis Platform(TCMSP),ETCM database and chemical database,and the target was converted into gene abbreviations in Uniprot database;Gene targets of PDPN were screened from Gene Cards database,Venny 2.1.0 was used to construct Venn diagram,and excel tables of intersecting gene targets of drugs and diseases were derived.Constructing "PPI" and "Drug-ingredient-Target" network diagrams using String database and Cytoscape3.9.1;The intersection targets of drugs and diseases were imported into DAVID database for enrichment analysis,and the mechanism of action of Yougui pills in the treatment of KOA was discussed.2 Preparation and grouping of KOA rat models:48 SPF SD rats(half male and half female)aged 3 weeks were selected for adaptive feeding for 7 days after receiving the animals.Then,48 SPF SD rats in this batch were randomly divided into four groups with 12 rats in each group according to the random number table method,and the males and females were separately fed in two cages in each group.The modified Hulth method was used to prepare the KOA rat model.The left knee joint of the rat was treated with anti-infection treatment for 3 days after operation,and the wound was expelled for 8 weeks after healing.3 KOA model identification: After the expulsion,one rat was randomly selected from the sham operation group and the model group,and its cartilage morphology was evaluated to evaluate whether the modeling was successful.4 Intervention methods: The dose of Yougui Pill group and celecoxib group was calculated according to the human-to-rat dose conversion formula,and the other groups were given conventional equal volume of distilled water for gavage.5 Experimental materials: after anesthesia of rats,blood was collected through abdominal aorta with a blood collection needle.After blood centrifugation,bleeding qing and plasma were separated and kept for later use.The knee cartilage was stripped and placed in EP tube and stored in ultra-low temperature refrigerator(-80℃)for future use.6 Morphological detection of rat chondrocytes: gross observation and HE staining were used to observe the pathological changes of rat cartilage.7 Observation and detection of chondrocyte indicators: The expression of p-Mek,Mek,p-Erk1/2,Erk1/2 and other proteins in rat cartilage tissue of each group were detected by Wsetern-blot,and the data of each group were analyzed and compared.Results:1 The main components of 1 Yougui Wan can reduce inflammatory response and affect chondrocyte decomposition to treat KOA by regulating AKT1,TNF,IL-6,IL-1B(IL-1β),VEGFA,TP53,CASP3 and other key targets.2 Yougui Pill mainly regulates MAPK signaling pathway,NF κ B signaling pathway and Wnt signaling pathway,and treats KOA by reducing the expression of inflammatory factors and slowing down the destruction rate of articular chondrocytes.3 Effects of Yougui Pill on serum IL-1β and TNFα contents of KOA rats: The levels of inflammatory factors such as IL-1β and TNFα in serum of rats in sham operation group were significantly lower than those in model group,which verified the success of KOA model construction.The expressions of IL-1β and TNF-α in serum of western medicine group and Yougui Wan group were significantly decreased,and the difference was statistically significant.4 Effects of Yougui pill on p-Mek,Mek,p-Erk1/2,Erk1/2 in chondrocytes: The expressions of p-Mek,Mek,p-Erk1/2 and Erk1/2 in the model group were significantly increased compared with the sham group.Compared with the model histone expression,Yougui Wan group and Xiyao group had a certain degree of decrease.Conclusion:1 The mechanism of Yougui Pill in the treatment of knee osteoarthritis is related to its MAPK signaling pathway reducing the expression of inflammatory factors and slowing down the destruction rate of articular chondrocytes.2 Yougui Wan can reduce the expression levels of IL-1β and TNFα inflammatory factors in serum of rats,and improve the inflammatory response.3 The mechanism of Yougui pill in the treatment of KOA is related to its regulation of MEK/ERK signaling pathway related factors.