| ObjectiveIn order to develop the basic of new anti-osteoporosis drugs,to explore the therapy of Gougujiao and Shuangxi Gujiaobao,and treatment-related pathways of osteoporosis disease,and to expand the research ideas and research direction of Traditional Chinese Medicine(TCM)in animal medicine.MethodsTheoretical research:Through combing the research on the pathomechanism,cause of disease and treatment of osteoporosis by the works and experts of TCM in the past,a comprehensive understanding of the characteristics of the treatment of osteoporosis by TCM,and a summary of the cognition and treatment ideas of TCM for the disease;The theory of "Complement form with form,Generation-inhibition in five elements" explores the theory of Gougujiao,and the theory of "Warming the kidney and assisting yang,Promoting blood circulation and promoting meridian" explores the theoretical basis of the Traditional Chinese Medicine treatment of Osteoporosis by the Shuangxi Gujiaobao,which provides sufficient theoretical basis for follow-up research.Experimental research:The component analysis and identification of Gougujiao and fried dog bone meal were carried out by metabolomics non-target analysis method combined with ultra-high phase liquid mass spectrometry,the differences of the two components were compared,and the advantages of processed drugs was summarize D.The superiority of the processing method of bone glue;applying estradiol valerate,Gougujiao and Shuangxi Gujiaobao to treat Retinoic acid induced osteoporosis in rats(model group),observe the basic body weight,rectal temperature,and auricle surface,comparison of temperature,visceral index,Bone Mineral Density,Bone Mineral Content,HE staining,MAS SON staining of the rats between the groups,analysis of the efficacy of Gougujiao and Shuangxi Gujiaobao on the model group;to test Serum phosphorus,calcium,and alkalinity urine phosphorus,calcium,by biochemical analyzer phosphate,applying ELISA method to test serum osteocalcin,Type ?procollagen N-terminal propeptide,Type ? collagen C-terminal peptide content,Gougujiao,Shuangxi Gujiaobao Bao on bone metabolism;test the serum OPG content by ELISA,and apply WB,PCR to detect the expression of RANK,RANKL,OPG,TRAF6,NF-?B at the protein and gene level,and use immunohistochemical method to observe and analyze the positive expression of RANKL and OPG,to explore Gougujiao,Shuangxi Gujiaobao anti-osteoporosis effect on the classic mechanism RANKL/OPG/TRAF6,analyze its therapeutic effect on the classic pathway RANK/RANKL/OPG and TRAF6/NF-?B signaling pathway,study the internalmechanism of Gougujiao and Shuangxi Gujiaobao how to anti-osteoporosis.ResultsTheoretical resultsOsteoporosis is in line with the concept of traditional Chinese medicine,the location of Osteoporosis in TCM is the foundation of kidney,related with the liver and spleen.It is rooted in the imbalance of Yin and Yang.The pathogenesis recorded in ancient documents is related to the loss of the kidney,such as kidney yin deficiency,kidney yang deficiency,and kidney yang deficiency.Insufficiency of kidney qi,in addition to spleen and kidney yang deficiency,liver and kidney dysfunction,etc.,over time,the disease progresses to kidney deficiency and blood stasis.Kidney essence deficiency,kidney qi deficiency and qi stagnation and blood stasis could be summarized as the fundamental pathogenesis of Osteoporosis;On the other hand,there are feelings of externalevil wind,cold and dampness,emotional disorders,congenital deficiencies,acquired disorders,excessive intercourse,five flavors inJures the viscera,and strains caused by the reserved personality.The treatment methods include warm and supplement kidney yang,enrich and nourish kidney yin,tonifying both yin and yang,treating the spleen and kidney simultaneously for nourishing phlegm-damp,and liver and kidney treatment for enriching and nourishing the liver and kidney.Based on the theory of"Complement form with form" and "Generation-inhibition in five elements" for the treatment of osteoporosis,Shuangxi Gujiaobao is based on the addition and subtraction of Zhongjing Shenqi Wan,the prescription applies the method of warming the kidney and strengthening the spleen,promoting blood circulation and clearing menstruation to treat osteoporosis caused by kidney deficiency and blood stasis.The compatibility of various prescriptions and Chinese medicines can enhance the effect of nourishing the kidney and nourishing bones,which provides an important theoretical basis for follow-up experimental research.Experimental resultsAmong the identifiable components,in the positive ion mode,212 different components were screened through Matching analysis,and 125 in the negative ion mode were screened and matched.In the positive ion mode,compared with fried dog bone powder,the relative expression levels of Gougujiao are relatively high components such as Aprobarbital,Choline,Betaine,Proline,L-phenylalanine,Nepetalactam,Newtrehalose,Phygrine,Hydroxyproline,etc.,including 6 kinds of amino acids;in negative ion Mode,the relatively high expression levels of Gougujiao are:Succinate,L-Proline,Adenine,Xanthine,Citric acid,Isokobusone,L-Norleucine,Myristic acid,Leucine acid,Adrenal acid,Hypoxanthine,Stearic acid,Tryptophan,etc.,including 4 amino acids.The bone mineral density and bone mineral content of the osteoporosis model rats modeled by retinoic acid were significantly reduced(P<0.001).The bone mineral density and bone mineral content of the osteoporosis model rats after treatment with estradiol valerate tablets,Gougujiao,and Shuangxi Gujiaobao The mineral content increased significantly(P<0.01,P<0.001);the HE staining results showed that the bone trabecular space of the osteoporosis model rats modeled by retinoic acid for increased(P<0.01),and the number of trabecular bones was slightly increased after the Shuangxi Gujiaobao treatment(P<0.01),the distance of the trabecular bone gap was significantly reduced after the treatment of the Shuangxi Gujiaobao drug(P<0.05).The serum ALP content of osteoporosis model rats modeled by retinoic acid for increased significantly(P<0.001),and the ALP content appeared after of treatment with estradiol valerate tablets,Gougujiao,and Shuangxi Gujiaobao(P<0.01,P<0.001).After of retinoic acid modeling,the serum calcium content of osteoporosis model rats decreased(P<0.05),and the serum calcium content increased after of retinoic acid modeling(P<0.05).The serum calcium content increased after of retinoic acid modeling(P<0.05).The serum phosphorus content of model rats was significantly increased(P<0.05),and the serum phosphorus content was significantly reduced after of drug treatment with estradiol valerate tablets and Gougujiao(P<0.05).After of treatment with ShuangxiGujiaobao,the urinary calcium content of osteoporosis model rats modeled by retinoic acid decreased significantly(P<0.01),and the urinary phosphorus content of model rats was significantly increased(P<0.05).after the treatment of estradiol valerate tablets,Gougujiao and Shuangxi Gujiaobao,the urinary phosphorus content decreased significantly(P<0.05,P<0.01).After of retinoic acid modeling,the serum PN1P content of osteoporosis model rats decreased(P<0.001),and after of drug treatment with estradiol valerate tablets,Gougujiao,and Shuangxi Gujiaobao,the PN1P content was somewhat different increased(P<0.05);after of retinoic acid modeling,the CTX-1 content of osteoporosis model rats increased(P<0.05),after of drug treatment of estradiol valerate tablets,Gougujiao,Shuangxi Gujiaobao,CTX-1 The content has decreased significantly(P<0.05,P<0.01);the osteocalcin content of osteoporosis model rats modeled by retinoic acid for has decreased significantly(P<0.01).After of treatment with estradiol valerate tablets and Shuangxi Gujiaobao,osteocalcin content is significantly increased(P<0.05,P<0.01).After of retinoic acid modeling osteoporosis model rats,the serum OPG content of osteoporosis model rats was significantly reduced(P<0.05);after of treatment with estradiol valerate tablets,Gougujiao,and Shuangxi Gujiaobao,the serum OPG content was significantly rise(P<0.01,P<0.001).The results of detecting tibial protein content by WB showed that the tibial RANKL,TRAF6,and NF-?B1 protein content of the osteoporosis model rats modeled by retinoic acid for increased significantly(P<0.01,P<0.05),and the OPG protein content decreased significantly(P<0.001);The RANKL protein concentration in the tibia of rats was significantly reduced after the drug treatment of estradiol valerate tablets,Gougujiao,and Shuangxi Gujiaobao(P<0.05);rats after of drug treatment with estradiol valerate tablets,Gougujiao,and Shuangxi Gujiaobao.The TRAF6 protein content of the tibia was significantly reduced(P<0.001),and the total OPG protein content of the tibia of rats was significantly increased(P<0.05);after of drug treatment with estradiol valerate tablets,Gougujiao,and Shuangxi Gujiaobao,the protein content of NF-?B1 in the tibia of rats was significantly reduced(P<0.05,P<0.01,P<0.001).The results of RT-PCR detection of tibia showed that the relative expression levels of RANK,RANKL,and TRAF6 genes in osteoporosis model rats modeled with retinoic acid for were significantly increased(P<0.01),and the relative expression levels of OPG genes were significantly reduced(P<0.05).After of drug treatment with estradiol valerate tablets,Gougujiao,Shuangxi Gujiaobao,the relative expression of rat RANK gene expression levels were significantly reduced(P<0.01,P<0.05);after of estradiol valerate tablets,Gougujiao,Shuangxi Gujiaobao,the relative expression of RANKL gene in rats was significantly reduced after treatment with drug(P<0.05);the relative expression of OPG gene was significantly increased after of drug treatment with estradiol valerate tablets,Gougujiao,and Shuangxi Gujiaobao(P<0.01,P<0.05);after The relative expression of TRAF6 gene was significantly reduced after drug treatment of estradiol valerate tablets,Gougujiao,and Shuangxi Gujiaobao(P<0.05).Immunohistochemical results showed that the positive expression of OPG in the femoral neck of the osteoporosis model rats modeled by retinoic acid for was significantly reduced(P<0.05).After of drug treatment with estradiol valerate tablets,Gougujiao,and Shuangxi Gujiaobao,the rat OPG the positive expression level was significantly increased(P<0.01,P<0.05);the positive expression level of RANKL in the femoral neck of the osteoporosis model rats modeled by retinoic acid for was significantly increased(P<0.001).After the drug treatment of estradiol valerate tablets,Gougujiao,Shuangxi Gujiaobao,the positive expression of RANKL in rats was significantly reduced(P<0.001).ConclusionsThe relatively high content of trehalose,matrine,and citric acid in Gougujiao are closely related to osteoporosis;The relative content of amino acids in Gougujiao is relatively high,among them,hydroxyproline and proline are the main ones,two amino acids It is closely related to the treatment of osteoporosis with Gougujiao;Gougujiao contains higher micellization components,succinate and stearic acid,which promotes the solubilization effect and promotes absorption by the body,while the higher content of group of Vitamin B in Gougujiao help maintain the stability of vitamins and promote the absorption of nutrients.Gougujiao and Shuangxi Gujiaobao can resist bone loss by adJusting bone density and bone metabolism.By regulating the RANKL/RANK/OPG signal pathway,it reduces the activation of osteoclasts,inhibits bone resorption and enhances bone formation.TRAF6 content inhibits the inflammatory response of NF-?B1 and inhibits the generation of osteoclasts,thereby achieving anti-osteoporosis effects by regulating the RANKL/OPG/TRAF6 signaling pathway. |
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