Mechanism Of Zuogui Pills On Osteoporosis In Ovariectomized Rats By Hepcidin-OPG/RANKL Pathway

ObjectiveThis study was carried out on the basis of preliminary work,using bone histomorphometry,bone biomechanics,immunohistochemistry,in situ hybridization and other experimental techniques,to verify the effect of Zuogui Pills on osteoporosis caused by ovariectomy,to further explore the changes in liver secretion of Hepcidin kidney deficiency and after nourishing kidney.The downstream OPG/RANKL pathway can be regulated from hepcidin to explore the mechanism of Zuogui Pills on osteoporosis.This will partially clarify the scientific connotation of the theory of"kidney being in charge of bone ",can be for traditional Chinese medicine treatment of osteoporosis to provide further experimental basis.MethodsSixty SPF female SD rats were randomly divided into three groups:blank group(n = 12),sham operation group(n = 12)and modeling group(n = 36).Rats in the modeling group were treated with bilateral ovariectomized(OVX)after anesthesia with pentobarbital sodium in a proportion of 45 mg/kg body weight.The rats in the sham operation group do not remove the ovaries,and only a few peripheral adipose tissue was removed around the ovary.Three months after surgery,the rats in the modeling group were divided into three groups:model group(OVX group),OVX +estradiol valerate group(E2 group),OVX + Zuogui pill group(Zuogui pill Group),each group of 12 rats.And then feeding drugs to rats of each group:giving water decoction of Zuogui pill to feeding the rats of Zuogui pill group,the concentration of 0.968 g crude drug/mL,the volume of the drug was | mL/100g body weight,The dose of 9.68 g crude drug/kg body weight;E2 group of rats given the estradiol valerate suspension,the concentration of 0.018 mg/mL,the volume of 1mL/100g body weight,The dose of 0.18 mg/kg body weight.The above gavage,once a day for 6 days,rest 1 day,and then continuous gavage of 6 days,continuous gavage for 3 months.The rats in the blank group,the sham operation group and the model group were given equal volume of pure water.During the period of gavage,the dose was adjusted according to the weight taken per week.Rats in each group were injected intraperitoneally with tetracycline hydrochloride(0.5ml/100g body weight)at 16 days and 3 days before sacrifice.The bone tissue was labeled with fluorescence.At the end of the gavage,the rats were sacrificed rapidly and take the liver and make paraffin sections,used to detect liver iron levels and hepcidin protein and mRNA expression.The left femur was used for the measuring of bone biomechanical indexes.The left tibia was made as non-decalcified bone slices for bone histomorphometric measurements,The left tibia was decalcification with EDTA,then making frozen sections,measuring the expression of Fpn1 and OPG,RANKL protein and mRNA in bone tissue.The experimental results are expressed as mean ± standard deviation(± s),SPSS 20.0 statistical software was used for statistical processing.First conduct a positive test,normal distribution data were compared between two or more groups,using single factor analysis of variance(ANOVA).Comparison between the two groups,the variance of the homogeneity of the LSD under the test.The variance is used under the Dunnett’s T3(3)test,if the data is non-normal distribution,the use of non-parametric test.Result1 Effects of Zuogui Pills on bone histomorphometric parameters in ovariectomized rats with osteoporosisThe TBV%of tibia in OVX group was significantly lower than that in sham operation group.The TBV%of the tibia in the E2 group and the Zuogui pill group was significantly higher than that in the OVX group,but still significantly lower than the sham operation group.Compared with sham group,the TRS%and TFS%of tibia were significantly increased in OVX group.The TRS%and TFS%of the tibia in the E2 group and the Zuogui pill group were significantly lower than those in the OVX group.However,there was no difference in TFS%of tibia between E2 group and sham operation group;but the TRS%and TFS%of the tibia and the TRS%in the E2 group were significantly higher than those in the sham operation group.The OSW,MAR and mAR of tibia in OVX group were significantly higher than those in sham operation group.Compared with OVX group,OSW,MAR and mAR of tibia were significantly decreased in E2 group and Zuogui pill group.There was no difference above between E2 group and sham operation group,but the OSW,MAR and mAR of Zuoguiwan group were significantly higher than those in sham operation group.2 Effects of Zuogui Pills on the Maximum Load(N),Flexural Strength(MPa)and Elastic Modulus(GPa)of OVX ratsThe maximal load,flexural strength and elastic modulus of femur in OVX group were significantly lower than those in sham operation group,compared with OVX group,the maximal load,flexural strength and elastic modulus of E2 group and Zuogui pill group were significantly increased,the flexural strength and elastic modulus of femur in E2 group were not different from those in sham operation group,so as the elastic modulus of femur with Zuogui pill group.However,the maximal load and flexion of the femur in Zuogui pill group and the maximal load of the femur in the E2 group were significantly lower than those in the sham operation group.3 Effects of Zuogui Pills on Iron Ion Level in Liver of OVX RatsCompared with the sham group,the percentage of Prussian blue coloring area in the OVX group was significantly improved,compared with OVX group,the percentage of Prussian blue coloring area in E2 group and Zuogui pill group decreased significantly,but still higher than the sham operation group.The results showed that the iron content of liver in OVX group was significantly higher than that in sham operation group,the iron content of liver in E2 group and Zuogui pill group was significantly lower than that in OVX group,but still higher than the sham operation group.4 Effects of Zuogui Pills on Expression of Hepcidin Protein and mRNA in Liver of OVX RatsCompared with the sham group,the expression of Hepcidin protein and mRNA in the liver of OVX group was significantly decreased,compared with OVX group,the expression of Hepcidin protein and mRNA in liver of E2 group and Zuogui pill group was significantly higher,but still significantly lower than the sham operation group5 Effects of Zuogui Pills on Expression of Fpnl,OPG,RANKL Protein and mRNA in Tibial Bone Marrow of OVX RatsCompared with the sham group,the expression of Fpn1 mRNA in the tibial bone marrow of OVX group was significantly higher,compared with OVX group,the expression of Fpnl mRNA in E2 and Zuogui pill group was significantly decreased,but still significantly higher than the sham operation group.Compared with the sham group,the expression of OPG protein and mRNA in the tibial bone marrow of OVX group was significantly decreased,compared with OVX group,the positive expression density of OPG protein and mRNA in E2 group and Zuogui pill group was significantly higher,there was no significant difference in OPG mRNA expression between IOD and sham group.The expression of OPG protein and mRNA in Zuogui pill group and OPG protein in E2 group were significantly lower than those in sham operation group.Compared with the sham group,the expression of RANKL protein and mRNA in the tibial bone marrow of OVX group was significantly higher,compared with OVX group,RANKL protein and mRNA expression of IOD in E2 group and Zuogui pill group were significantly decreased,but still higher than the sham operation group.There was no significant difference between the sham operation group and the blank control group,which excluded the effect of surgical factors on the experimental results.Conclusions(1)Surgical resection of rat ovaries can lead to osteoporosis with high-bone-turnover activity,which bone resorption and bone formation are both increased,but because bone resorption is greater than bone formation,resulting in loss of bone mass.While the Zuogui pills can reduce the high bone turnover rate,improve bone mass,for ovariectomy osteoporosis has effect.(2)Surgical removal of rat ovaries can lead to liver secretion of Hepcidin reduction,iron ion deposition,iron overload occurs.While RANKL expression on bone marrow cells was significantly up-regulated,OPG and Fpnl expression were significantly down-regulated.The two together,resulting in abnormal osteoclasts active,the two factors leading to abnormal osteoclasts and bone loss.Zuogui pill can improve the secretion of Hepcidin levels,improve iron overload due to ovariectomy,and then adjust the downstream OPG/RANKL signal path,eventually reducing osteoclast activity,reduce bone loss.This is one of the mechanisms by which it can treat osteoporosis.The above results show that the regulation of Hepcidin on iron overload plays an important role in the "kidney being in charge of bone".