| With the acceleration of aging,more and more people suffer from two or three or even multiple diseases at the same time,a phenomenon named "comorbidity".Studies have reported that the incidence of comorbidities began to climb since 1988.From 2013 to 2014,60%of the US population had two or more comorbidities,nearly 40%had three or more comorbidities,and 23%had four or more comorbidities,and the incidence increased with age.Osteoporosis and osteoarthritis are common comorbidities of body diseases.Osteoporosis(OP)is a systemic bone disease with reduced bone mass,degeneration of bone microstructure and increase of bone brittleness,which can easily lead to fracture.Osteoarthritis(OA)is also a common clinical disease of bone and joint.The pathological changes are characterized by degeneration and destruction of articular cartilage,hyperplasia and hardening of bone.Osteoporosis and osteoarthritis are more common in older women,and one of the most important pathologic factors is the decline of estrogen in the body with age.Based on the previous research experience of the research group,this study used female rats to remove ovaries and simulate the pathological state of osteoporosis comorbidities Osteoarthritis to observe the efficacy of electroacupuncture intervention.ObjectiveTo observe the effect of electroacupuncture on subchondral bone and cartilage in rats w ith osteoporosis and osteoarthritis which induced by ovariectomized,as well as the influence of RANKL/OPG signaling pathway.Methods90 3-month-old female clean grade Sprague-Dawley rats were randomly divided into three groups:sham operation group,ovariectomized(OVX)group and electroacupuncture(EA)group,with 30 rats in each group.In the OVX and EA groups,bilateral ovaries were removed,while in the sham-operated group,only adipose tissue of the same size as the ovary was removed.Rats in the EA group were treated with electroacupuncture at the acupoints of Shenshu(BL23),Taixi(KI3),Xuehai(SP10).Zusanli(ST36)and Yanglingquan(SP9)on both sides.Density wave(dispersing wave frequency 3Hz,dense wave frequency 15Hz)was used,and the intensity was measured by slight muscle contraction(0.7-1.0mA,Once a day,30 minutes each time,5 days a week;The sham operation group and the OVX group did not intervene.At the 4th,8th and 12th weeks after operation,10 rats in each group were randomly killed.Experiment 1:Histopathology detection:Morphology of articular cartilage was observed by SaffronO-Fast Green Staining and Mankin score was performed to evaluate the effect of EA on knee cartilage in rats.Experiment 2:Imaging detection:The subchondral bone(BV/TV,Tb.Th,Tb.N,Tb.Sp)was analyzed by Micro-CT,to evaluate the effect of EA on subchondral bone of knee joint in rats.Experiment 3:Biochemical detection:Urinary C-terminal cross-linking telopeptide of type I collagen(UCTX-I),Urinary C-terminal cross-linking telopeptide of type II collagen(UCTX-II)and Serum 17β-oestradiol(E2)were determined to evaluate the effects of EA on bone resorption,cartilage degradation and estrogen in subchondral bone of knee joint in rats by enzymy-linked immunosorbency assay(ELISA).Experiment 4:Molecular biological detection:The mRNA expression levels of Matrix Metallo Proteinase 13(MMP13),osteoprotegerin(OPG)and receptor activator of NF-KB ligand(RANKL)were detected by RT-PCR.Tthe protein expression levels of MMP13,OPG and RANKL were detected by Western-blot to evaluate Changes in MMP13 and in the RANKL/OPG signaling pathway.ResultsExperiment 1:Histopathology detection results showed that EA inhibits degeneration of knee cartilage in rats.At the 4th,8th,12th weeks,Mankin score of OVX group was higher than that of sham operation group(P<0.05),the differences were statistically significant.At the 4th,8th weeks,there was nosignificant difference between EA group and OVX group(P>0.05).At the 12th week,Mankin score of EA group was lower than that of OVX group(P<0.05),the differences were statistically significant.Experiment 2:Imaging detection results showed that EA inhibits subchondral osteoporosis.BV/TV and Tb.Th:At the 4th,8th,12th weeks,BV/TV and Tb.Th of OVX group was lower than that of sham operation group(P<0.05),while that of EA group was higher than that of OVX group(P<0.05),the differences were statistically significant.TB.N:At the 4th,8th,12th weeks,TB.N of OVX group was lower than that of sham operation group(P<0.05),the differences were statistically significant.The EA group was higher than the OVX group,the difference was not statistically significant at the 4th week(P>0.05),but the difference was statistically significant at the 8th and 12th week(P<0.05).Tb.Sp:At the 4th,8th,12th weeks,Tb.Sp of OVX group was higher than that of sham operation group(P<0.05),while that of EA group was lower than that of OVX group(P<0.05),the differences were statistically significant.Experiment 3:Biochemical detection results showed that EA inhibited subchondral bone resorption,cartilage degeneration and the decline of estrogen.UCTX-Ⅰ:At the 4th,8th,12th weeks,UCTX-Ⅰ in OVX group was higher than that in sham operation group(P<0.05),the differences were statistically significant.UCTX-Ⅰ in EA group was lower than that in OVX group(P<0.05),the differences were statistically significant.UCTX-Ⅱ:UCTX-Ⅱ in the 4th week after operation,the OVX group was higher than the sham operation group(P>0.05),the EA group was lower than the OVX group(P>0.05),the differences were not statistically significant.However,on the 8th and 12th week after surgery.the OVX group was higher than the sham operation group(P<0.05),and the EA group was lower than the OVX group(P<0.05),the differences were statistically significant.E2:At the 4th,8th,12th weeks,E2 of OVX group was lower than that of sham operation group(P<0.05),while that of EA group was higher than that of OVX group(P<0.05),the differences were statistically significant.Experiment 4:Molecular biological detection results indicated that EA inhibits cartilage degeneration and subchondral osteoporosis of knee joint in rats.MMP13 mRNA:At the 4th week,there was no significant difference in MMP13 mRNA expression among the three groups(P>0.05).At the 8th week,the expression of MMP13 mRNA in the OVX group was higher than that in the sham-operated group,and the EA group was lower than that in the OVX group,but the difference was not statistically significant(P>0.05).At the 12th week,the expression of MMP13 mRNA in the OVX group was higher than that in the sham-operated group(P<0.01),and that in the EA group was lower than that in the OVX group(P<0.01),the differences were statistically significant.OPG mRNA:At the 4th,8th and 12th week after operation,OPG in the OVX group was lower than that in the sham-operated group(P<0.01,P<0.01,P<0.01).and that in the EA group was higher than that in the OVX group(P<0.01,P<0.01,P<0.01),with statistical significance.RANKL mRNA:At the 4th,12th weeks,the OVX group was higher than the sham-operated group(P<0.01,P<0.01),and the EA group was lower than the OVX group(P<0.01,P<0.05),the differences were statistically significant.At the 8th week,the OVX group was higher than the sham-operated group(P<0.01),and the EA group was lower than the OVX group(P<0.01),the differences were statistically significant.At the 12th weeks,the expression of MMP13 protein in the OVX group was higher than that in the sham-operated group(P<0.01),the differences were statistically significant,while the EA group was lower than that in the OVX group(P>0.05),the difference was not statistically significant.The expression of OPG protein in the OVX group was lower than that in the sham-operated group(P<0.01),and the EA group was higher than that in the OVX group(P<0.01),the differences were statistically significant.The expression of RANKL protein in OVX group was higher than the sham-operated group(P<0.01),and the EA group was lower than the OVX group(P<0.05),the differences were statistically significant.ConclusionIt was found that electroacupuncture may improve knee subchondral osteoporosis and inhibit knee cartilage degeneration by regulating RANKL/OPG signaling pathway,suggesting that EA is an effective method for the treatment ofosteoporosis and osteoarthritis comorbidities,which provides a theoretical basis for electroacupuncture treatment of osteoporosis and osteoarthritis comorbidities. |
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