| ObjetiveAccording to the research that invigorating spleen and tonifying kidney plus reinforcing bone acupuncture method can improve the antiseptic effect of femur and oxidative stress of bone tissue in postmenopausal osteoporosis model(PMOP)rats,to explore the regulation mechanism and effect target of PMOP by invigorating spleen and tonifying kidney plus reinforcing bone acupuncture method from further studies that the regulation of FoxO/Wnt-β-catenin signaling pathway mediated osteoblast differentiation and bone formation.Method(1)Animal experiment:Grouping and modeling:68 6-month-old SPF female SD rats(weighing 220-250g)were randomly divided into two groups by computer:Sham group(n=13)and OVX group(N=55).In the sham group,part of the fat tissue around the ovary was removed,and in the model group,both ovaries were removed(castration).Three months after surgery,a total of 65 rats(12 in the Sham group and 53 in the model group)survived in the two groups.Two rats in the Sham group and three rats in the OVX group were randomly selected.Bone Density(BMD)and Bone microstructure were detected by dual-energy X-ray and light microscopy,indicating that the PMOP model was established.50 PMOP model rats were randomly divided into 5 groups,including model group(OVX group)(n=10),medicine group(Med group)(n=10),acupuncture group(Acu group)(n=10),acupuncture combined with medicine group(Med+Acu group)(n=10)and estradiol group(E2 group).Treatment methods:Sham group and OVX group were given normal saline gavage,0.5ml/100g,once a day,for 6 months;Med group was given alendronate sodium intragastric administration.dissolved 70mg/tablet in 100mL normal saline,9*kg ml intragastric administration,the dosage was adjusted according to body weight,once a week,for 6 months;Acu group was treated with invigorating spleen and tonifying kidney plus reinforcing bone acupuncture method at Zu Sanli,Shenshu and Dazhu acupoints,giving needling manipulation every 10 minutes and pull out the needles after 20 minutes,three times a week,for 6 months;Med+Acu group was treated with alendronate sodium gavage plus invigorating spleen and tonifying kidney plus reinforcing bone acupuncture method.The dosage was the same as the Med group,and the treatment course and frequency were the same as the Acu group;E2 group was given diethylstilbestrol by gavage,and the diethylstilbestrol was put into distilled water and prepared into a suspension concentration of 4.5μg/ml,0.5ml/100g for each dose,once a day,for 6 months.The weight of the rats was weighed weekly,and the dose was adjusted according to the weight change.Detection of indicators:Sample collection:After the last intragastric administration,abdominal aorta puncture was used to collect blood and centrifuge serum;The rats were sacrificed and bilateral intact femurs were removed,the right femur was fixed with 4%paraformaldehyde solution,and the left femur was placed in liquid nitrogen storage tank at-196℃ and preserved.The detection indicators are as follows:1.Efficacy indicators:①BMD test:BMD of left femur(whole segment of femur)in rats was measured by dual-energy X-ray bone density instrument;② Bone microstructure:all parameters of bone microstructure were detected by Micro-CT;③ Mechanical properties:the minimum external force of isolated femur fracture was detected by the stable minimum external force experimental device of femur fracture made by the previous project;④ Bone histomorphology:Ultra-thin sections of femur in sagittal plane were stained by HE,and the density,diameter of adipocytes and osteocytes,the distribution and arrangement of bone trabeculae were observed under microscope.2.Mechanism Indicators:① Serum estrogen level:The serum estrogen level in each group was detected by ELISA.② Bone metabolism indexes:The contents of serum bone calcium(BGP)and osteosin(OPG)were determined by ELISA.③ Oxidative stress index:Serum glutathione(GSH),superoxide dismutase(SOD)and glutathione peroxidase(GPX)were detected by ELISA.④ Signaling pathway indicators:The protein levels of FoxO3a,β-catenin,Wnt2,P-SHC and SHC in bone tissues were detected by Western blot,and the gray values of the bands were used to quantify the protein levels.⑤the mRNA expressions of PPAR-y,Runx2 and OPG in bone tissue were detected by real-time PCR.(2)Clinical study:Grouping:According to the diagnostic criteria of PMOP,patients were recruited from the Department of Orthopedics and Rehabilitation of the First Affiliated Hospital of Dali University and Yunnan Hospital of Traditional Chinese Medicine through sample size calculation,and 72 patients meeting the inclusion criteria were divided into control group and treatment group by "Computer pseudo-random number generation method ".Treatment:The control group was given alendronate sodium orally,10mg/time,once a day,for 6 months;Acupuncture group was treated with integral adjustment acupuncture with Dazhu,Shenshu and Zu Sanli as main points,adding and subtracting points according to syndrome differentiation,twice a week for 6 months on the basis of the treatment of the control group.Detection of indicators:General information:patient name,age,years of menopause,body mass index,waist circumference;Efficacy and efficacy evaluation indexes:①Bone mineral density test:L1-4BMD was detected by DEXA bone density instrument;②Visual Analogue Scale(VAS):Visual Analogue score was used to examine the pain degree of patients;③ TCM syndrome score:TCM syndrome score table was used to check the symptom score of patients;3.Mechanism indicators:① Serum estrogen:Serum E2 was detected by ELISA;② Bone metabolism indicators:serum β-CTX,TRACP,BGP,BNALP were determined by ELISA;③Oxidative stress index:Serum AOPP,T-AOC,SOD and MAOA were detected by ELISA.4.Safety indicators:Patients’ blood pressure,pulse,respiration,temperature,blood,urine,fecal routine,liver and kidney function,electrocardiogram and adverse reactions were monitored during treatment.Result(1)Animal experiment:①Determination of modeling results:Compared with the Sham group,the body weight of the three randomly selected rats in the OVX group was significantly increased,and the BMD was 36.51%lower than that in the Sham group.The trabecular bone was sparse and decreased,and the trabecular bone was significantly thinner and wider,indicating the establishment of OP model.② Efficacy indicators:6 months after treatment,compared with other 5 groups,BMD of OVX group was significantly reduced and the trabecular three-dimensional network structure was abnormal,the bone volume fraction,structural model index,trabecular volume,trabecular width,trabecular number and trabecular thickness decreased significantly,the trabecular spacing increased,and the minimum external force of femur fracture in vitro decreased significantly,with statistical significance(P<0.05).The results showed that BMD,bone microstructure and mechanical properties were decreased after 6 months of castration.There was no significant difference in the above indexes between Acu group,Med group and E2 group compared with the sham operation group(P>0.05),but there was significant difference between Acu group and OVX group(P<0.05),indicating that acupuncture,alendronate sodium and acupuncture and drug combination can prevent osteoporosis,and acupuncture combing drug can synergistically increase the curative effect.③Mechanism indicators:Compared with the other 5 groups,E2 level in OVX group decreased significantly,BGP level increased,OPG level decreased,SOD,GPX,GSH level decreased,β-catenin,Wnt2 expression decreased,FoxO3a expression increased,Runx2,OPG mRNA expression decreased.The mRNA expression of PPAR-y2 was significantly increased(P<0.05).There was no significant difference between Acu group,Med group,E2 group and Sham group(P>0.05),but significant difference between Acu group and Med+Acu group(P<0.05),indicating that acupuncture,alendronate sodium and estrogen can regulate the expression of Wnt-β-catenin pathway related proteins.Regulate the state of oxidative stress in the body,and effectively resist oxidative stress,in which the combination of acupuncture and medicine can synergistic effect.(2)Clinical study:① General data:there were no statistically significant differences in age,body mass index,menopausal years and waist circumference between the two groups(P>0.05),indicating comparability.② Efficacy indicators:compared with the control group,bone mineral density in acupuncture group was higher,and the difference was statistically significant(P<0.05);Compared with the control group,VAS score and TCM syndrome score of acupuncture group were significantly decreased(P<0.05),indicating that acupuncture can effectively increase bone mineral density and relieve pain and other symptoms caused by osteoporosis.③Mechanism indicators:Compared with the control group,the level of E2 in the treatment group was higher,and the difference was statistically significant(P<0.05).Compared with the control group,the levels of β-CTX,TRACP and BNALP in the treatment group decreased,the levels of BGP increased,the levels of AOPP and MAOA decreased,the levels of TAOC and SOD increased(P<0.05),indicating that acupuncture can effectively improve bone metabolism and prevent and cure PMOP through anti-oxidative stress.(4)Safety indicators:During the treatment,all patients had normal liver and kidney functions,blood routine,stool routine and urine routine,and normal basic vital signs(blood pressure,heart rate,respiration and temperature).During treatment,no serious adverse reactions were found in 2 groups.There were 1 cases of nausea and 1 case of constipation in the control group,2 cases of dizziness and 2 cases of constipation in the treatment group,all of which were cured without special treatment.Conclusions(1)Animal experimental studies suggest that:①invigorating spleen and tonifying kidney plus reinforcing bone acupuncture method can improve bone mineral density,bone microstructure,mechanical properties of femur,regulate bone metabolism,increase osteogenic differentiation and bone formation,and effectively prevent and treat PMOP;② The mechanism of invigorating spleen and tonifying kidney plus reinforcing bone acupuncture method to prevent and cure PMOP may be:Via improving estrogen level,anti-oxidative stress,improving bone metabolism microenvironment,cross-regulating FoxO/Wnt-β-catenin pathway,inhibiting FoxO transcription in oxidative stress,up-regulating Wnt expression,regulating high conversion level of bone metabolism,promoting bone formation and inhibiting bone resorption in model rats.(2)The clinical results showed that:① Acupuncture combined with conventional Western medicine had better efficacy on PMOP than western medicine alone.②Acupuncture is a safe and effective treatment for PMOP patients by increasing serum estrogen level and regulating oxidative stress state in vivo,thereby reversing the postmenopausal high transition state of bone metabolism,promoting bone formation and reducing bone absorption... |
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