| Osteoarthritis is a disease that involves the entire joint structure,and the damage of cartilage and subchondral bone is the most important manifestation of osteoarthritis.Clinical studies have confirmed that Gubi Zhitongfang(GBZTF)has a good effect on the treatment of osteoarthritis,but the specific mechanism of action has not been clarified.Objective: In this paper,we are going to use data mining technology to analyze the pattern of GBZTF formula;confirm that GBZTF can effectively alleviate the symptoms of osteoarthritis and improve the microenvironment of subchondral bone through in animal experiments;and investigate the biomolecular mechanism of GBZTF in regulating the microenvironment of subchondral bone by using the MC3T3-E1 cell assay.Methods:1 Retrieve the literature of TCM for OA in the database.Use TCMISSV2.5 software to carry out statistics and screen the literature to analyze the four qi,five flavors,attributes,and the pattern of grouping of clinical TCM prescriptions for the treatment of OA,and make comparisons with GBZTF.2 6-week-old SD male rats were used to establish the sham-operation group,DMM model,and GBZTF low,medium,and high dose groups,in which the sham-operation group and model group were gavaged with pure water,and the GBZTF dose groups were gavaged.After 8 weeks of continuous gavage,Micro-CT was used to observe the changes in the cartilage and subchondral bone of the knee joint of rats,Elisa assay was used to analyze the serum-related inflammatory indexes of rats,and RT-q PCR was used to analyze the expression of bone metabolism-related genes in the articular cartilage as well as the expression of genes related to the osteogenic differentiation of the subchondral bone tissue.3 Effects of different concentrations of GBZTF on the activity of MC3T3-E1 cells as observed by CCK-8;the effects of GBZTF on the differentiation of MC3T3-E1 cells were observed by ALP staining;the inhibitory effects of GBZTF on the mineralization of MC3T3-E1 cells were observed by ARS staining;and Western Blot and RT-q PCR were used to establish that MC3T3-E1 regulates the molecular biological mechanism of osteoblasts.Results:1 Through analysis,it was found that the most frequently occurring drugs in the prescription were deficiency tonic drugs,with a total of 46 flavors and 2,584 occurrences,among which the common drugs were hyssop,angelica sinensis,and ripened dihuang;followed by blood circulation and blood stasis activators,with a total of 38 flavors and 1,489 occurrences,including chuanxiong ligustici,chickweed vine,and boneset,which were commonly found in the prescription.Among these two classes of drugs,Radix Rehmanniae Praeparata appeared in the Bone Paralysis and Pain Relief Formula as the king’s medicine,and Rhizoma Chuanxiong,Rhizoma Polygonati Odorati,Rhizoma Polygonati Odorati appeared in the formula as the subject’s medicine.Statistical analysis of the four qi,five flavors,and meridians of the drugs showed that the frequencies of warm,neutral,cold,hot,and cool drugs were 4369,1769,828,146,and 111;and the frequencies of pungent,bitter,sweet,sour,salty,and astringent drugs were 4218,3624,3084,697,407,and 98 in that order;The top 5 drugs belong to the meridians of liver,kidney,spleen,heart and lung in order;among them,the frequency of belonging to the liver meridian could be 4957 times,the frequency of kidney meridian 2801 times,the frequency of spleen meridian 2730 times,the frequency of heart meridian 2501 times and the frequency of lung meridian 1264 times.Through the statistics of taste and meridian,it was found that the six medicines in GBZTF were all warm;meanwhile,the sweet medicines possessed 5 flavors,3 bitter,1 pungent,and 1 salty;the medicines in GBZTF were mainly attributed to the liver and kidney meridians,among which 6 flavors were attributed to the liver meridian,5 flavors to the kidney meridian,and 1 flavor to the large intestine meridian.To summarize,the whole formula has the effect of tonifying the liver and kidney,relaxing the tendons and bones,dispelling wind and relieving pain,and is suitable for OA patients with deficiency of the liver and kidney.2 GBZTF in medium and high doses can reduce the abnormal growth of nerve in subchondral bone at the early stage of arthritis,effectively relieve pain and improve joint movement in arthritic rats.GBZTF can effectively alleviate the degeneration of knee joint cartilage by promoting cartilage anabolism and inhibiting catabolism;it can inhibit the abnormal osteoclastogenesis by regulating the expression of osteoblastic genes,and improve the microenvironment of subchondral bone.3 GBZTF effectively inhibited the proliferation,differentiation and mineralization of MC3T3-E1 cells.RT-q PCR results suggested that GBZTF could inhibit the expression of the osteogenic differentiation genes Osx,Opn,Col1a1 and Ocn,and the Western Blot results showed that the inhibitory effect of GBZTF on osteoblasts’ proliferation and differentiation was achieved through the inhibition of activation of TGF-β1/PI3K/Runx2,AKT/Runx2,AKT/Runx2,AKT/Runx2,AKT/Runx2 and AKT/Runx2.AKT/Runx2 signaling pathway.Conclusion:The data mining analysis revealed that the commonly used drugs for treating OA in clinical practice were deficiency tonic drugs,the four qi of the drugs were mainly warm and neutral,the taste of the drugs were mainly pungent,sweet and bitter,and the drug meridians were mainly the liver and kidney meridians.The results of analyzing the association of GBZTF provide a reliable theoretical basis for clinical treatment.The in vivo and in vitro experiments revealed that GBZTF could effectively alleviate the symptoms of arthritis and improve the microenvironment of subchondral bone;it also significantly inhibited the proliferation,differentiation and mineralization of MC3T3-E1 cells;GBZTF could be involved in the regulation of the expression of genes related to the differentiation of osteoblasts to regulate the microenvironment of subchondral bone,which was realized by inhibiting the activation of the TGF-β1/PI3K/AKT/Runx2 signaling pathway.signaling pathway activation.These findings deepen the understanding of the regulatory role of GBZTF in OA and provide new ideas for the prevention and treatment of osteoarthritis-related diseases. |
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