| ObjectiveIn this study,ovariectomy(OVX)rats were used as the OP model of kidney deficiency.Based on Hedgehog signaling pathway,the therapeutic effect and mechanism of Zuogui Pill on OVX osteoporosis rats were discussed.In order to further clarify the mechanism of "kidney governing bone" and supply rationable evidence for the treatment of osteoporosis by traditional Chinese medicine.MethodsThree-month-old SD rats(n=78)were divided into 3 groups at random according to their body weight after adaptive feeding,including the blank control group(n=12),the sham operation group(n=15)and the model group(n=45).Bilateral ovaries were removed in the model group to prepare the op rat model of "kidney deficiency".In the sham operation group,the ovaries were not removed,and a small amount of adipose tissue around the ovary was removed,and the other operation processes were the same as those in the model group.Nine rats died due to anesthesia and operation.The number of rats in the sham operation group was reduced to 12.After 12 weeks of castration,the model group was randomly divided into three groups according to body weight:ovariectomy group(OVX group,n=13),OVX+estrogen group(positive control group,n=13)and OVX+Zuogui Pill group(Zuogui Pill group,n=13).After grouping,the rats in each administration group were administered by gavage once a day for six consecutive days and rested for one day.The drug was administered for 12 weeks.The weight of rats was measured every week to adjust the dosage.After administration,the rats in each group were killed immediately after anesthesia.The histomorphological indexes of femoral bone were detected by CT.The protein and mRNA expressions of OPG,RANKL,Ihh,Ptch,Smo and Glil in tibial bone marrow were detected by immunohistochemistry and in situ hybridization.Results1 Effect of Zuogui Pill on BMD of femur in ovariectomized rats Results in comparison with sham operation group,there was a significant decline of BMD in the OVX group,positive control group and Zuogui Pill group(P<0.01);Compared with OVX group,the BMD in E and Zuogui Pill group were significantly higher(P<0.01,P<0.05).2 Effect of Zuogui Pill on histomorphological indexes of femoral bone in ovariectomized rats2.1 Effect of Zuogui Pill on BV/TV of femur in ovariectomized ratsResults in comparison with sham operation group,the BV/TV value of femur in OVX group declined significantly(P<0.01);Compared with OVX group,the BV/TV value in positive control group and Zuoguiwan group had a remarkable improvement(P<0.01),but it was still significantly lower than that in sham operation group(P<0.01).2.2 Effect of Zuogui Pill on SMI of femur in ovariectomized ratsThe SMI of femur in OVX group was higher than that in sham operation group(P<0.01);The femoral SMI in positive control group and Zuogui Pill group had a marked drop than that in OVX group(P<0.01,P<0.05),but evidently higher than that in sham operation group(P<0.01).2.3 Effect of Zuogui Pill on Tb.Th in femur of ovariectomized ratsThere was no statistical difference of the femur Tb.Th of rats in each group(P>0.05).2.4 Effect of Zuogui Pill on Tb.N in femur of ovariectomized ratsOVX group,positive control group,Zuogui Pill group femoral Tb.N had a significant decline than that in sham operation group(P<0.01);positive control group and Zuogui Pill group femur Tb.N was higher than that in OVX group(P<0.01).2.5 Effect of Zuogui Pill on Tb.Sp in femur of ovariectomized ratsCompared with the sham operation group,Tb.Sp in OVX group increased evidently(P<0.01);positive control group and Zuogui Pill group Tb.Sp decreased evidently than the OVX(P<0.01),but it was still significantly improve than that in the sham operation group(P<0.01).3 Effect of Zuogui Pill on the expression of key factors of Hedgehog signal pathway Ihh,Ptch,Smo and Gli1 in tibial bone marrow of ovariectomized rats3.1 Effect of Zuogui Pill on Ihh mRNA and protein expression in tibial bone marrow of ovariectomized ratsThe expression levels of Ihh mRNA and protein in tibial bone marrow of OVX group,positive control group and Zuogui Pill group were significantly higher than those in sham operation group(P<0.01,P<0.05);Compared with OVX group,the expression levels of Ihh mRNA and protein in tibial bone marrow of rats in positive control group and Zuogui Pill group decreased significantly(P<0.01)3.2 Effect of Zuogui Pill on the expression of Ptch mRNA and protein in tibial bone marrow of ovariectomized ratsThe expression levels of Ptch mRNA and protein in tibial bone marrow of OVX group were significantly higher than those in sham operation group(P<0.01);The expression levels of Ptch mRNA and protein in tibial bone marrow of rats in positive control group and Zuogui Pill group were significantly lower than those in OVX group(P<0.01),but still significantly higher than those in sham operation group(P<0.05).3.3 Effect of Zuogui Pill on the expression of Smo mRNA and protein in tibial bone marrow of ovariectomized ratsThe expression levels of Smo mRNA and protein in tibial bone marrow of OVX group were significantly higher than those of sham operation group(P<0.01);The expression levels of Smo mRNA and protein in tibial bone marrow of rats in positive control group and Zuogui Pill group were significantly lower than those in OVX group(P<0.01),but the expression levels in Zuogui Pill group were still evidently higher than those in sham operation group(P<0.05),and there was no difference between positive control group and sham operation group(P>0.05).3.4 Effect of Zuogui Pill on the expression of Gli1 mRNA and protein expression in tibial bone marrow of ovariectomized ratsThe expression levels of Gli1 mRNA and protein in tibial bone marrow of OVX group were evidently higher than the sham operation group,(P<0.01).Compared with OVX group,the expression levels of Gli1 mRNA and protein in tibial bone marrow of positive control group and Zuogui Pill group were lower(P<0.01),but still significantly higher than that of sham operation group(P<0.05).4 Effect of Zuogui Pill on the expression of OPG,RANKL mRNA and protein expression and RANKL/OPG value in tibial bone marrow of ovariectomized rats4.1 Effects of Zuogui Pill on the expression of OPG,RANKL mRNA and RANKL/OPG in tibial bone marrow of ovariectomized ratsThe expression level of OPG mRNA in tibial bone marrow of OVX group decreased significantly(P<0.01)than the sham operation group,,the expression level of RANKL mRNA increased significantly(P<0.01),and the ratio of RANKL/OPG increased significantly;The expression level of OPG mRNA in tibial bone marrow of rats in positive control group and Zuogui Pill group was significantly higher than that in OVX group(P<0.01),but it was still lower than that in sham operation group(P<0.05,P<0.01);The expression level of RANKL mRNA was remarkable lower than that in OVX group(P<0.01),and the ratio of RANKL/OPG was significantly lower(P<0.01),but it was still significantly higher than that in sham operation group(P<0.05,P<0.01).4.2 Effects of Zuogui Pill on the expression of OPG,RANKL protein and RANKL/OPG in tibial bone marrow of ovariectomized ratsCompared with the sham operation group,the expression level of OPG protein in tibial bone marrow of OVX group decreased significantly(P<0.01),the expression level of RANKL protein increased significantly(P<0.01),and the ratio of RANKL/OPG increased significantly.The expression level of OPG protein in tibial bone marrow of rats in positive control group and Zuogui Pill group was significantly higher than that in OVX group(P<0.01),but it was still significantly lower in Zuogui Pill group than that in sham operation group(P<0.05),and there was no difference between positive control group and sham operation group(P>0.05);The expression level of RANKL protein was significantly lower than that in OVX group(P<0.01),and the ratio of RANKL/OPG was significantly lower(P<0.01),but it was still significantly higher than that in sham operation group(P<0.01).ConclusionOvariectomy for 6 months can cause obvious osteoporosis in rats.After ovariectomy,the mRNA and protein expression levels of key factors Ihh,Ptch,Smo and Gli1 of Hedgehog signaling pathway increased significantly,and Hedgehog signaling pathway was abnormally active;Thus,the ratio of RANKL/OPG increased,the activity of osteoclasts increased abnormally,resulting in bone loss and osteoporosis.Zuogui Pill has obvious therapeutic effect on osteoporosis in ovariectomized rats.Zuogui Pill can inhibit the abnormal activity of Hedgehog signal pathway caused by ovariectomy,and significantly reduce the expression levels of key factors Ihh,Ptch,Smo,Gli1 mRNA and protein;Then reduce the ratio of RANKL/OPG,inhibit the activity of osteoclasts and prevent bone loss.These results suggest that Hedgehog signaling pathway plays an important role in the process of "kidney governing bones". |
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