Study On The Effect And Mechanism Of Gancao Fuzi Decoction On Osteoarthritis In Rats

Objective:In this study,HPLC and UHPLC-QE-MS were used to qualitatively and quantitatively analyze the chemical constituents in the aqueous extract of Gancao Fuzi decoction.The rat model of osteoarthritis was created by MIA knee cavity injection,and Gancao Fuzi decoction was administered intragastrically to investigate the prevention and treatment effect and mechanism of licorice aconite decoction on osteoarthritis in rats.MIA was used to damage primary chondrocytes,and drug containing serum of Gancao Fuzi decoction was administered to investigate the protective effect and mechanism of licorice aconite decoction on inflammatory chondrocytes.Method1.Qualitative and quantitative analysis of the chemical components in the aqueous extract of Gancao Fuzi decoction were carried out by HPLC and UHPLC-QE-MS.2.The rat model of osteoarthritis was established by intraarticular injection of sodium iodoacetate（MIA）.After modeling,normal group and model group were given normal saline,positive group was given glucosamine sulfate,and Gancao Fuzi decoction low-dose,medium-dose and high-dose groups were given 3 g·kg^-1,6 g·kg^-1and 12 g·kg^-1,respectively.All animals were given continuous administration for 30 days.Animal behavior tests were performed once a week during administration.After sampling,ELISA kit was used to detect the contents of related biochemical indexes in the lavage fluid and serum.He staining was used to observe the pathological state of the joint.WB method was used to detect the expression of related proteins in cartilage.3.The articular cartilage protein was extracted and detected by Nano-LC-LTQ-Orbitrap system.Protein Discovery software for Protein identification,Sieve software for relative quantitative analysis of proteins,Panther Classification System database for String analysis of differential proteins and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment signaling pathways.4.The serum containing Gancao Fuzi decoction was prepared by the method of Chinese medicine serum pharmacology.The primary chondrocytes of fetal rats were extracted.After 2h stimulation by MIA,the drug-containing serum of Gancao Fuzi decoction was cultured for 48h.The proliferation of chondrocytes was detected by CCK-8 method;ELISA kit was used to detect the content of related biochemical indexes in medium supernatant;WB assay was used to detect the expression of related proteins in chondrocytes;Apoptosis of chondrocytes was observed by FITC-Annexin V/PI fluorescence double staining;The expression of MMP-3 in chondrocytes was observed by ICC.To investigate the protective effect of serum containing glycyrrhiza aconite decoction on chondrocytes,and block the PI3K pathway of chondrocytes with blocker LY294002,so as to preliminarily explore the protective mechanism of glycyrrhiza aconite decoction on chondrocytes.Results1.Through detection and data analysis,489 compounds were identified in the decoction of licorice aconite decoction,including 104 flavonoids,55 phenylpropanoids,107 terpenoids,7 coumarins and their derivatives,45 alkaloids,38 phenols,and 133 other compounds.2.The results showed that the pain index and joint width of rats were significantly increased after MIA modeling.The contents of TNF-α,IL-1βand MMP-3 in Guanjianjian lavage fluid were significantly increased,while the content of TIMP-1 was significantly decreased;The articular surface of rats was rough and damaged,the cartilage layer became thin,the synovial hyperplasia thickness was uneven,the joint space became larger,and the chondrocytes were arranged disorderly;The protein expression of Bax and MMP-3 in cartilage tissue was up-regulated,while the protein expression of Bcl2 was down-regulated.After administration,the pain index and joint width of rats were significantly improved;The contents of MMP-3,TNF-αand IL-1βin lavage fluid were decreased,while the content of TIMP-1 was increased;Improve joint injury,make joint surface become smooth,joint space becomes smaller,reduce inflammation of synovial tissue;It can significantly down-regulate the expression of Bax and MMP-3 in cartilage tissue,and up-regulate the expression of Bcl-2 in cartilage tissue.3.3.Proteomics analysis showed that there were 273 differential proteins in each group.KEGG analysis showed that these differential proteins were involved in 43 signaling pathways,including Systemic lupus erythematosus,11 of which were associated with osteoarthritis.4.The survival rate of chondrocytes induced by 9μm MIA for 2 hours was 50%to 70%,which could be used as the modeling condition for subsequent experiments.After MIA stimulation,the contents of MDA,TNF-α,IL-1βand MMP-3 in the medium increased,SOD activity and TIMP-1content decreased significantly,and the protein expressions of MMP-3 and Bax in chondrocytes were up-regulated.After administration of serum containing Gancao Fuzi decoction,the inhibitory effect of MIA on chondrocyte proliferation was significantly improved,the contents of MDA, TNF-α,IL-1βand MMP-3 in the medium were decreased,and SOD activity and TIMP-1 content were increased.The expression of MMP-3 and Bax protein in chondrocytes was decreased,and the ratio of intracellular Bcl-2/Bax was increased to improve cell apoptosis.After LY294002 was added to block the PI3K/Akt signaling pathway,the MMP-3 content increased,while the MMP-3 content decreased after administration of the drug-containing serum of Glycyrrhiza aconite decoction.Conclusion1.There are a lot of chemical components in the Gancao Fuzi decoction.A total of 489 compounds were identified by means of liquid-mass spectrometry,which were mainly flavonoids,terpenoids,coumarins and their derivatives,phenylpropanoids,etc.Glycyrrhizin,glycyrrhizic acid,cinnamic acid and coumarin can be separated by HPLC under certain conditions.2.Gancao Fuzi decoction can improve the pain of osteoarthritis and joint swelling in rats,and has a protective effect on articular cartilage tissue by reducing the content of reactive oxygen species in rats,reducing the content of inflammatory factors in articular cavity,inhibiting the apoptosis of chondrocytes.3.Proteomics was used to screen out the different proteins in each group,and the function of the different proteins and the signal pathways involved were discussed,suggesting that Gancao Fuzi decoction may achieve the effect of treating osteoarthritis by regulating the protein expression of inflammatory and apoptotic pathways such as PI3K-Akt signal pathway and HIF-1 signal pathway.4.The drug-containing serum of Gancao Fuzi decoction can promote the proliferation of chondrocytes,reduce the level of oxidative stress and inflammation of chondrocytes,inhibit the apoptosis of chondrocytes,and have a protective effect on the inhibition of chondrocyte function by MIA,the mechanism may be through the regulation of the expression of PI3K-Akt signaling pathway related proteins.