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Study On The Clinical And Mechanism Of Wenshen Tongluo Zhitong Decoction In The Treatment Of Senile Osteoporosis

Posted on:2024-01-21Degree:DoctorType:Dissertation
Country:ChinaCandidate:T Y GuanFull Text:PDF
GTID:1524307202950869Subject:Fractures of TCM science
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Objective:To observe the therapeutic effect of Wenshen Tongluo Zhitong Decoction on senility osteoporosis,explore the influence of Wenshen Tongluo Zhitong Decoction on senility bone marrow mesenchymal stem cells,and clarify the specific mechanism of Wenshen Tongluo Zhitong Decoction against senility osteoporosis by regulating bone marrow mesenchymal stem cells.Methods:(1)A total of 96 patients with senile osteoporosis treated from December 2022.6-February 2019 were selected as the study objects,randomly grouped according to the random number method,and the closed envelope method was used for grouping concealance.These patients were divided into control group(n=48)and observation group(n=48).The control group received basic treatment(calcium carbonate D3)+alendronate sodium tablets,and the observation group:Basic treatment(calcium carbonate D3)+Wenshen Tongluo Zhitong Decoction.Serum of patients before and after medication was collected,PTH,N-MID,TRACP,SOD and FBLN1 levels were detected by ELISA,bone mineral density was detected by QCT,clinical efficacy.TCM syndrome score,VAS,COQOL score,FRAX fracture risk assessment,muscle strength grading,cognitive function assessment,bone metabolism index and bone mineral density of the two groups were compared,and drug safety was observed and evaluated.(2)Male C57BL/6J mice were selected to construct the experimental model,and were randomly divided into aging model group(D-gal group),research group(low,medium and high dose groups of Chinese medicine Wenshentongluo Zhitong Decoction),control group and alenaphine sodium group by random number method.The senile osteoporosis mouse model was prepared by intrabitoneal injection of D-galactose.Serum levels of bone calmodulin-related protein(OPG)andβ-collagen crosslinking(β-CTx)were detected by ELISA,and histopathological changes of tibia were observed by HE staining.Bone mineral density(BMD),bone volume fraction(BV/TV),trabecular number(TB.N),trabecular thickness(TB.Th)and trabecular separation(TB.Sp)were measured by micro-CT.(3)Four-week-old SAMR1 mice were selected to obtain primary bone marrow mesenchymal stem cells by whole bone marrow method,and the cells were identified by flow cytometry,immunofluorescence and osteogenic adipogenic differentiation.D-galactose was successfully used to induce senescent bone marrow mesenchymal stem cells,and the senescence process was interfered with with the drug-containing serum of Wenshen Tongluo Zhitong Decoction.The aging of bone marrow mesenchymal stem cells was detected by β-galactosidase cell aging staining,the osteogenic differentiation of bone marrow mesenchymal stem cells was detected by ALP staining and alizarin red staining,the lipogenic differentiation of bone marrow mesenchymal stem cells was detected by oil red O staining,and the ROS in bone marrow mesenchymal stem cells were detected by flow cytometry and immunofluorescence staining.The contents of MDA and SOD in the supernatant of bone marrow mesenchymal stem cells were detected by ELISA.(4)D-galactose was used to induce senescent bone marrow mesenchymal stem cell model,and Wenshen Tongluo Zhitong Decoction drug-containing serum and AMPK inhibitor were used to intervene the senescence process.The aging of bone marrow mesenchymal stem cells was detected by β-galactosidase cell aging staining,the osteogenic differentiation of bone marrow mesenchymal stem cells was detected by ALP staining and alizarin red staining,the lipogenic differentiation of bone marrow mesenchymal stem cells was detected by oil red O staining,and the ROS in bone marrow mesenchymal stem cells was detected by flow cytometry and immunofluorescence staining.The contents of MDA and SOD in the supernatant of bone marrow mesenchymal stem cells were detected by ELISA,and the expressions of LC3B and Beclinl in autophagy were detected by immunofluorescence.D-gal was used to construct a mouse model of senile osteoporosis,and the related proteins of aging and AMPK/mTOR autophagy were detected by Western Blotting.Results:(1)ELISA results showed that compared with before treatment,PTH and FBLN1 levels were significantly up-regulated and TRACP levels were significantly down-regulated in the study group after treatment,but N-MID and SOD were not significantly different.There was no significant difference in TCM syndrome score between the study group and the control group before treatment.After treatment,the TCM syndrome score of the study group was significantly different from that of the control group.There was no significant difference in BMD between the study group and the control group before treatment.After treatment,the bone mineral density of the study group was significantly different from that of the control group.The total effective rate of the study group was 93.33%,which was significantly better than that of the control group 88.89%.(2)ELISA results showed that the expression of serum bone metabolism markers OPG and βCTX could be effectively inhibited by Wenshen Tongluo Zhitong Decoction in senil-induced osteoporosis mice.The results of H&E staining showed that the number of bone trabeculae increased,the width and continuity of bone trabeculae increased in SOP mouse model.Micro-CT results also showed that Tb.N,Tb.Th,BMD,BV/TV,Tb.Pf were significantly increased,and Tb.Sp was significantly decreased in the compound group.(3)Flow cytometry,immunofluorescence,and osteogenic lipid differentiation results showed that the extracted mouse bone marrow mesenchymal stem cells were consistent with the biological characteristics of animal derived bone marrow mesenchymal stem cells,SA-β-gal staining results showed that warming kidney and Tongluo and analgesia could alleviate D-galactose-induced cell senescence.The results of ALP and alisarin red staining showed that Wenshen Tongluo Zhitong Decoction could promote bone formation and differentiation of bone marrow mesenchymal stem cells.The results of oil red 0 staining showed that Wenshen Tongluo Zhitong Decoction could inhibit lipid-forming and differentiation of bone marrow mesenchymal stem cells.The results of fluorescence quantification and flow cytometry showed that Wenshen Tongluo Zhitong Decoction could reduce the cellular oxidative stress reaction.ELISA results showed that Wenshen Tongluo Zhitong Decoction had the ability of anti-oxidative damage.(4)The results of Western Blotting showed that warming the kidney to Tongluo analgesia could alleviate the D-galactose-induced cell senescence.Quantitative analysis by Western Blotting and immunofluorescence showed that warming the kidney to Tongluo analgesia could alleviate the decrease of intracellular autophagy caused by D-galactose.The results of Western Blotting showed that warming kidney and Tongluo analgesia could regulate AMPK/mTOR signaling pathway in BMSC to activate autophagy.Both SA-β-gal staining and Western Blotting showed that inhibition of AMPK-mTOR signaling pathway could inhibit the alleviating effect of Wenshen Tongluo Zhitong Decoction on galactose-induced cell senescence.The results of ALP and alizarin red staining showed that Wenshen Tongluo Zhitong Decoction could promote osteogenic differentiation of bone marrow mesenchymal stem cells,while AMPK inhibitor could reverse the effect.The results of oil red O staining showed that Wenshen Tongluo Zhitong Decoction could inhibit the lipogenic differentiation of bone marrow mesenchymal stem cells,while AMPK inhibitor reversed the effect.ELISA results showed that inhibition of AMPK-mTOR signaling pathway could inhibit the effect of Wenshen Tongluo Zhitong Decoction on oxidative damage induced by galactose.Western Blotting and immunofluorescence results showed that inhibition of AMPK-mTOR signaling pathway could inhibit the relieving effect of Wenshen Tongluo Zhitong Decoction on galactose-induced oxidative stress.In vitro experiments showed that Wenshen Tongluitong could significantly reduce the expression of aging markers P16 and P53 in femur of model group,and up-regulate the expression of autophagy related proteins Beclin-1 and LC3B,and increase p-AMPK/AMPK ratio and decrease p-mTOR/mTOR ratio.Conclusion:Wenshen Tongluo Zhitong Decoction has remarkable curative effect on senile osteoporosis,which can effectively improve the syndrome of traditional Chinese medicine,bone metabolism and increase bone density.Wenshen Tongluo Zhitong Decoction can increase bone density,improve bone microstructure,delay the senescence of bone marrow mesenchymal stem cells and promote bone formation in senile osteoporosis mice.The anti-osteoporosis mechanism of Wenshen Tongluzhitong prescription may be related to the regulation of AMPK/mTOR signaling pathway of bone marrow mesenchymal stem cells and the activation of autophagy to exert bone protection..
Keywords/Search Tags:Wenshen Tongluo Zhitong Decoction, Senile osteoporosis, Autophagy, AMPK/mTOR, Aging, Bone marrow mesenchymal stem cells
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