Discussion On The Therapeutic Effects And Mechanism Of Zuogui Pill On Osteoporosis Of Ovariectomized Rats Based On Hepcidin

ObjectiveThe present study was to explore the therapeutic mechanism thatthe Zuogui pill treat osteoporosis using bone mineral density detection, Micro CT, immunohistochemistry, enzyme-linkedimmunosorbentassay (ELISA) and other experimental techniques on the rat osteoporosis model, from the aspect of hepcidin maintain iron homeostasis. Hope to get further to know the pathogenesis of "The kidney governing the bones" and finally provide experimental foundation for clinical meditation of adopting Chinese meditation to treat osteoporosis.Methods60 female SD rats were selected and allocated to three groups randomly:12 in normal control group,12 in sham operation group and 36 in bilateral ovaliectomizedgroup. After anesthesia, both ovaries of all the rats in the bilateral ovaliectomizedgroup were extirpated. After three months of the operation, the rats of the bilateral ovaliectomizedgroup were divided into 3 sub-groups again:12 in modelgroup (OVX group),12 in OVX with estradiol valerate group (E2 group) and 12 in OVX with Zuogui pilltreated group (Zuogui pill group). The Zuogui pill group rats were administrated orally Zuogui pill decoction with the concentration of 0.968 g/mL and the E2 group rats were administrated orally estradiol valerate solution with the concentration of 0.018 g/mL. The above mentioned medicated volume is 1 mL/100g body weight. The medicated volume shall be adjusted according to the weight. The drug were given once a day, six days continuously then one day pause, the whole period lasted 3 months. The rats in other two groups were fed with the same volume diluted water. After Medication, the ratswere anesthetized toget artery blood. The blood was placed quietly 1 hour, and then centrifuged at 3000r/min,20min to get serum. The serum was isolated and conserved at -80℃. We centrifuged to detect thelevel ofVariouscytokinesin serum ELISA. After the rats being killed, the spiner were taken for bone mineral density testing; the right femur of the rats were retained to detect the tone tissue morphology; at the sametime, the right tibia of the rats were used for frozen tissue sectionafterbeingdecalcified to observe theproteinexpressionof Fpnl bymeans ofimmunohistochemistry.The experimental results data is expressed by Mean±SE (x±s) by adopting SPSS20.0 statistical software. If the data is normal distribution, the ANOVA shall be adopted to conduct data processing. The LSD test shall be adopted for the comparison between grouts at the time of homogeneity of variance; the Dunnett’s T3(3) test shall be adopted at the time of heterogeneity of variance; if the data is not normal distribution, the non-parametric test shall be adopted on Kruskal-Wallis single factor ANOVA (K sample) (W).Results1 Effects of Zuogui pill on the uterus coefficient of ovariectomized osteoporosis ratCompared with sham operation group, the rats’uterus coefficient in OVX group, E2 group and Zuogui pill group all decreased significantly.Compared with OVX group, the rats’uterus coefficient in E2 group increased significantly, while the Zuogui pill group showed no significant difference.2 Effects of Zuogui pill on lumbar vertebra bone mineral density of ovariectomized osteoporosis ratCompared withsham operation group, the lumbar BMD of rats in OVX group decreased significantly.Compared with OVX group, the lumbar BMD of rats in E2 group and Zuogui pill group increased significantly, while those parameter in E2 group and Zuogui pill group are significant lower thansham operation group.3 Effects of Zuogui pill on indexes of bone histomorphometry of ovariectomized osteoporosis rat3.1 Effects of Zuogui pill on femur BV/TV in ovariectomized ratsComparedwithsham operation group, the femur BV/TV of rats in OVX group decreased significantly.Compared with OVX group, the femur BV/TV of rats in E2 group, Zuogui pill group increased significantly, while those parameter in E2 group and Zuogui pillgroup are significant lower thansham operation group.3.2 Effects of Zuogui pill on femur Tb.N, Tb.Th, Tb.Sp in ovariectomized ratsCompared withsham operation group, the rats’ Tb.N in OVX group, E2 group and Zuogui pill group decreased significantly, while the rats’ Tb.Sp increased significantly.Compared with OVX group, the rats’ Tb.N in E2 group and Zuogui pill group increased significantly, while the rats’ Tb.Sp decreased significantly.The rats’ Tb.Th in every group showed no significant difference.3.3 Effects of Zuogui pill on femur SMI in ovariectomized ratsCompared withsham operation group, the rats’ SMI in OVX group increased significantly.Compared with OVX group, the rats’ SMI in E2 group and Zuogui pill group decreased significantly, while those parameter in E2 group and Zuogui pill group significant higher thansham operation group.4 Effects of Zuogui pill on serum Ferritin, Hepcidin, BGP, COL Ⅰ and TRACP-5b levels in ovariectomized rats4.1 Effects of Zuogui pill on serum Ferritin and Hepcidin levels in ovariectomized ratsCompared withsham operation group, the rats’ serum Ferritin in Zuogui pill group increased significantly, while the Hepcidin decreased significantly.Compared with OVX group, the rats’ serum Ferritin in E2 group and Zuogui pill group decreased significantly, while the Hepcidin increased significantly, there still have difference with sham operation group.4.2 Effects of Zuogui pill on serum BGP and COL Ⅰ levels in ovariectomized ratsCompared with sham operation group, the rats’ blood serum BGP and COL Ⅰ in Zuogui pill group are both increased significantly.Compared with OVX group, the rats’ serum BGP and COL Ⅰ in E2 group and Zuogui pill group decreased significantly.Compared with sham operation group, the rats’serum COL I in Zuogui pill group increased significantly.4.3 Effects of Zuogui pill on serum TRACP-5b level in ovariectomized ratsCompared with sham operation group, the rats’ serum TRACP-5b in OVX group increased significantly.Compared with OVX group, the rats’ serum TRACP-5b in E2 group and Zuogui pill group decreased significantly.4.4 Effects of Zuogui pill on tibia expression of Fpnl in bone marrow of ovariectomized osteoporosis ratCompared with sham operation group, the protein expression IOD of Fpnl in bone marrow in OVX group increased significantly.Compared with OVX group, the protein expression IOD of Fpn1 in bone marrow in E2 group and Zuogui pill group decreased significantly.The protein expression IOD of Fpnl in bone marrow in Zuogui pill group still significant higher than Sham operation group. There has no significant difference on the protein expression IOD of Fpnl in bone marrow between E2 group and Zuogui pill group.Conclusions(1) Ovariectomy can cause rat osteoporosis which is characterized by low bone mass, microarchitectural deterioration of bone tissue, while Zuogui pill can increase the bone mineral density of ovariectomized rats, accelerate the formation of bone and the good change of the bone microstructure(2) The levels of serum BGP, COL and TRACP-5b wererose and bone mass was lost after the rat was ovariectomized, while Zuogui pill canretrieve the phenomena to cure osteoporosis.(3) Ovariectomy can cause the serum hepcidin level decreased,which can lead to iron overloaded; while Zuogui pill can increase the serum hepcidin level and retrieve the iron content.In summary, the iron homeostasis maintained by hepcidin plays an important role in the the pathogenesis of "The kidney governing the bones".