The Effect Of Serum From Electro-acupunctured OP Rats On The MRNA And Protein Expression Of OPG And RANKL In Osteoblasts

ObjectiveBy observing the effects of acupunctured rats serum on the m RNA and protein expression of OPG and RANKL in rat calvarial osteoblasts(ROBs),to study the influence of acupuncture on the bone metabolism pathway OPG/RANKL,and provide theoretical base for the acupuncture treatment of osteoporosis.Methods60 two-month female SPF SD rats were divided into five group randomly,in which three groups were used the surgery to cut off the bilateral ovariectomy and then named the group of electroacupuncture,the group of western medicine,the group of model.The other two groups were blank group which were doing nothing for them and the group of sham operated control which cut and exposed the bilateral ovariectomy instead of removing.After the three months when the rats were raised in the laboratory of SPF by the common way,they were detected and the results said that the models were done.The western medicine group rats were administrated estradiol valerate by means of intragastrical administration for 3 courses(0.02mg/ml,1ml/100 g,5 day altogether).The electroacupuncture stimulation was applied to“Pishu”(BL 20),“Shenshu”(BL 23),“Sanyinjiao”(SP 6)and “Zusanli”(ST 36)for 30 min,5day altogether,3 courses with the same volume distilled water.The model group rats and sham operated control group rats were administrated distilled water for 3 courses.The blank group rats were not treated.After the three courses,detecting the bone mineral density and the bone mineral content was to evaluate the efficacy.Then the all group rats were anesthetized by the chloral hydrate and hemospasia from heart.Every group of blood sample was colded on the level of 4℃ for one hour、centrifugated(2500r/min,25min)、and dealed with 56℃ water bath and 0.22 um filter mesh.Osteoblast was derived from ten SD rat primary which were born within 2 days,by splitting skull and collagenase digestion.Primary osteoblast was cultured for24 hours,adding the serum by the ratio of 10%(the volum of serum/the volume of osteoblast culture medium=1/10).After 3 days,4% paraformaldehyde fixed the cell slide(containing1/1000 DEPC in paraformaldehyde in situ hybridization)The mRNA of OPG and RANKL were detected by means of immunofluorescence cytochemistry.The protein of OPG andRANKL were detected by means of immunofluorescence cytochemistry.Results1.After three months when the SD rats were cut off the bilateral ovariectomy,the result of bone mineral density said that the contain of bone mineral density and bone mineral content in model group were lower than the blank group(P<0.01).It proved that the model of osteoporosis was done.After three courses of treatment,the treatment of acupuncture and western medicine do increase the contain of bone mineral density and bone mineral content.Compared with the model group,there was significantly difference(P<0.01).The result said that the efficacy of acupuncture was better than the western medicine.2.Results of OPG in situ hybridization: Compared with the blank group,the expression of OPG mRNA of model group was definitely low(P<0.01).Compared with the sham operated control group,the expression of OPG mRNA of model group was definitely low(P<0.01).After the treatment of acupuncture and western medicine,it was increased.Compared with the model group,there was statistically significant(P<0.01).3.Results of RANKL in situ hybridization:Compared with the blank group,the expression of RANKL mRNA of model group was definitely increased(P<0.01).Compared with the sham operated control group,the expression of RANKL mRNA of model group was definitely increased(P<0.01).After the treatment of acupuncture and western medicine,it was decreased.Compared with the model group,there was statistically significant(P<0.01).4.Results of OPG immunohistochemical staining: Compared with the blank group,the expression of OPG protein of model group was definitely low(P<0.01).Compared with the sham operated control group,the expression of OPG protein of model group was definitely low(P<0.01).After the treatment of acupuncture and western medicine,it was increased.Compared with the model group,there was statistically significant(P<0.01).5.Results of RANKL immunohistochemical staining: Compared with the blank group,the expression of RANKL protein of model group was definitely increased(P<0.01).Compared with the sham operated control group,the expression of RANKL protein of model group was definitely increased(P<0.01).After the treatment of acupuncture and western medicine,it was decreased.Compared with the model group,there was statistically significant(P<0.01).ConclusionThe treatment of acupuncture significantly improved the bone mineral density,and the serum from acupunctured rats significantly increased the OPG mRNA and protein expression.In addition,the serum from acupunctured rats dramatically decreased the mRNA and protein expression of RANKL.The current research suggests that the treatment of acupuncture can adjust the protein and mRNA expression of the OPG and RANKL on osteoporosis rats.In other words,acupuncture treatment can regulate the bone formation and the bone absorption of the rats through OPG/RANKL /RANK pathway,and as a results,osteoporosis is prevented.