Study On The Drug Rules Of Master Of National Medicine,Bai Ling Liu In Treating Knee Osteoarthritis And The Mechanisms Of Action Of Critical Couplet Medicines

Objective: To compile National TCM Master Bailing Liu guidelines for treating Knee Osteoarthritis(KOA)and identify the molecular mechanisms of important drug pairings.Methods: 1.Compile the historical information of 277 cases of treatment of KOA(Qi Stagnation and Blood Stasis Syndrome)from June 2012 to December 2019,entered into the "Chinese medicine inheritance assisted platform(V2.5)" for summary analysis,and identify key drug pairs by collecting paper medical records of visitors of Professor to the National Hospital of Jilin Province.2.The dose of the drug for mice was calculated using the animal experimental dose conversion method and prepared frozen dried powder of Spatholobi Caulis and Rhizoma Drynariae(SR);the experimental animals were divided into five groups: the Sham group,the DMM group,the 23mg/ml group,the 46mg/ml group,and the 92mg/ml group,respectively.A knee osteoarthritis(KOA)mouse model was established using the DMM method.The knee joint cycle was measured and recorded for 0,7,14,and 28 days prior to and following modeling.Lower limb loads and step characteristics of the mouse were recorded weekly after modeling.A micro-CT scan detected red-green cartilage in the mouse joint after SR intervention.A q RT-PCR analysis determined the synthesis and degradation of metabolic marker gene expression in the SR post-intervention joint cartilage.Lastly,immunomodulatory factors were assessed.3.Alcian coloring was utilized to distinguish primary cartilage cells extracted from C57BL/6 mice from which cartilages were derived.Implement Cell Counting Kit-8(CCK-8)to ascertain the ideal concentration and timing of SR intervention cells;Whitexin-1β(IL-1β)to construct a model of cellular inflammation;DCFH-DA probe to quantify the concentration of Reactive Oxygen Species(ROS)in SR-intervention cells;Alcian chromometry to evaluate the protective effect of SR on protein polysaccharide in a cartilaginous extracelular matrix;4.The molecule mechanism of SR treatment for KOA is predicted through the utilization of network pharmacology and molecular coupling techniques.Subsequently,in vitro experimental validation is conducted.Results:1.Through data mining on 277 prescriptions and 128 traditional Chinese medicines,the top 14 most frequently used Chinese medicines were Myrrh,Achyranthes Bidentata,Angelica sinensis,Earthly Dragon,Chuanxiong,Licorice,Gentiana macrophylla,Peach Kernel,Wulingzhi,Xiangfu,Honghua,Qianghuo,Spatholobi Caulis and Rhizoma Drynariae.The four qi of medicine mainly consists of warming and calming;The five flavors are mainly bitter and spicy;The meridians mainly involve the liver,spleen,heart,and kidneys.Through the rules of formula composition and drug clustering analysis,it was found that myrrh and Achyranthes bidentata;Safflower,myrrh;Chicken Blood Vine,Bone Shredded Bu;Myrrh,chicken blood vine;Myrrh,bone fragmented tonic;Ox knee,safflower,these six drug combinations are the most commonly used.Based on association rule analysis,six drug pairs were obtained and preliminarily screened through CCK-8experiment.It was found that SR had the best intervention effect on chondrocytes.Therefore,SR was selected to treat KOA and explore its molecular mechanism of protecting chondrocytes and improving joint cartilage injury.2.In vivo experiments,according to the animal experiment dosage conversion method,the drug doses were determined to be 23mg/ml,46mg/ml,and 92mg/ml for in vivo experiments.The DMM method was used to establish a KOA rat model,and the knee joint circumference was observed before and after modeling.There was no significant difference in knee joint circumference among the groups before modeling.After 28 days of modeling,the DMM group showed significant swelling,while the swelling decreased after SR intervention.After 28 days of SR intervention,the joint circumference of the SR intervention group decreased significantly,which was significantly different from that of the DMM group.After 28 days of modeling,the results of the differences in leg weight bearing,force area,and standing time showed that the difference in the DMM group was significantly greater than that in the Sham group,and the difference decreased after medication intervention.The Micro CT results showed that the joint surface of the Sham group was flat and smooth,while the DMM group had significant osteophyte formation and cartilage defects.Different doses of SR group had less osteophyte formation and a smooth cartilage contour.Analyzing the bone trabecular volume and total volume fraction(BV/TV),trabecular thickness(Tb.Th),and trabecular separation(Tb.Sp)of subchondral bone,relative to the DMM group,after SR intervention,BV/TV,Tb Th increases,Tb Sp decreases.Using safranin green staining to detect the status of rat joint cartilage after SR intervention and scoring,the results showed a significant increase in the number of normal chondrocytes.The q RT-PCR results showed that compared with the DMM group,the expression levels of Acan and Comp in the joint cartilage increased after SR intervention.The immunohistochemical staining results showed that the expression of Col2a1 protein increased and the expression of MMP13 protein decreased in the joint cartilage after SR intervention.3.In vitro experiments were conducted to extract chondrocytes from C57BL/6 mice,and Alcian staining was used to identify the chondrocytes,revealing a dark blue color.The CCK-8 results showed that after 24 hours of SR intervention,concentrations of 0.1mg/ml,0.2mg/ml,and 0.4mg/ml were the most effective.Using IL-1β Establishing a cellular inflammation model,Alcian staining results showed that IL-1 β After induction,the number of cells decreased,but after intervention with SR,the number increased.Detection of ROS levels in cells using DCFH-DA probe method,quantitative display using flow cytometry,IL-1 β After induction,the ROS content decreased,and SR inhibited the decreasing trend.q RT-PCR was used to detect the levels of synthetic and catabolic biomarkers in chondrocytes after SR intervention,and the results showed IL-1β After induction,the expression of MMP13 increased,while the expression of Col2a1 and Comp decreased.SR intervention reduced the expression of MMP13 and promoted the expression of Col2a1 and Comp.4.34 active ingredients in SR were predicted through network pharmacology.The visualization network of drug component target disease includes 211 nodes and 619 edges.There are 114 common targets between drug component targets and disease targets.A protein-protein interaction(PPI)network was constructed,which includes 114 nodes and1971 edges.The average Degree value of the nodes was 34.6,and the clustering correlation was 0.684.The expected value of the edges was 626,and the P-value of protein action was<1.0e-16.The top 20 target proteins were screened for GO and KEGG analysis,resulting in 20 biological,cellular,and molecular functions.The predicted biological processes(BP)mainly involve responsetoxinobiotic stimuli,responsetooxygenlevels,regulation of apoptotic signaling pathways,etc.The predicted molecular functions(MF)mainly involve oxidoreductase activity,cytokineceptorbinding,antagonistic activity,etc.The cell composition(CC)mainly involves cytokines such as membrane,vesiclelumen,organelleouter membrane,and endoplasmicreticulum.Through KEGG pathway analysis,it mainly involves IL-17 signaling pathway,Osteoclastdifferentiation,NF kappa B signaling pathway,MAPK signaling pathway,etc.Seven key compounds identified through screening,Luteolin,beta sitosterol,Formonetin,Kaempferol,8-o-Methylreusi,Vestitol,and naringenin,were docked with the key protein IL-17 in the IL-17 signaling pathway.The results showed that the binding energies were-7.2,-7.0,-6.7,-7.0,-6.8,and-7.1,respectively,with good binding energies.Detection of IL-17 and NF-κB in articular cartilage after SR intervention.The gene and protein expression levels of and Cola1 showed that IL-17 and NF-κB in the DMM group expression increases,Col2a1 expression decreases,IL-17 and NF-κB after SR intervention expression decreases while Col2a1 expression increases.Conclusion: 1.Through data mining and experience summary of Professor Bailing Liu treatment of KOA(Qi Stagnation and Blood Stasis Syndrome),it provides useful reference for the treatment of KOA(Qi Stagnation and Blood Stasis Syndrome)in theory,while screening core drugs for SR,providing theoretical support for clinical medication.2.In vivo experiments have shown that SR can inhibit IL-1β production The induced decrease in the content of proteoglycans and type II collagen in the extracellular matrix of chondrocytes promotes synthetic metabolism,inhibits catabolism,and reduces ROS content to alleviate oxidative damage,maintaining the normal function of chondrocytes,thereby exerting a protective effect.3.In vitro experiments have shown that SR reduces joint swelling,prolongs standing time,increases foot support and load-bearing area,thereby increasing hind limb coordination and improving walking patterns in bone KOA rats.SR promotes an increase in the content of proteoglycans,maintains the normal state of chondrocytes,promotes the expression of the synthetic metabolic marker Col2a1 in cartilage,and reduces the expression of the catabolic metabolic marker MMP13.SR can improve the subchondral bone state,reduce osteophyte formation and cartilage defects.4.Network pharmacology prediction found that SR may exert its effect by inhibiting the IL-17 signaling pathway.Molecular docking between the main components of the drug and key targets of the IL-17 pathway showed that the binding energies were all less than-0.5,indicating good binding energy.Verified through in vitro experiments,key IL-17 and its downstream NF-κB after SR intervention The expression levels of gene and protein decreased,while the expression of Col2a1 increased.SR inhibits IL-17/NF-κB.The signaling pathway plays an anti-inflammatory,antioxidant,and inhibitory role in ECM degradation,thereby protecting chondrocytes,reducing osteophyte formation,and improving KOA symptoms.SR can inhibit the IL-17/NF-κB signaling pathway and exert anti-inflammatory,antioxidant,and ECM degradation inhibition effects,thereby protecting chondrocytes,reducing bone mineral formation,and improving KOA symptoms.