| Purpose:Based on the TCM typing characteristics of kidney essence deficiency and bone marrow loss in patients with primary osteoporosis,Zuogui Pill,a classical prescription for tonifying kidney and nourishing Yin,was selected to investigate the effect of serum containing Zuogui Pill and ER pathway on osteoclast differentiation and osteoclast-related inflammatory factors in RANKL-stimulated RAW264.7 macrophages.To reveal the mechanism of inhibiting bone resorption and provide experimental basis for Zuogui Pill in preventing and treating primary osteoporosis.Material and method:35 SPF male SD rats（240270 g）were fed adaptively for 7days.According to their body weight,they were randomly divided into five groups:the control group,Zuogui Pill low dose group,Zuogui Pill medium dose group,Zuogui Pill high dose group and Progynova group,with 7 rats in each group.The clinical doses of Zuogui Pill in rats at low,medium and high doses were 4.84 g·kg-1,9.68 g·kg-1,19.36 g·kg-1.The clinical dosages of Progynova in rats was 0.09 mg·kg-1.The control group was given normal saline(10 ml·kg-1),once a day for 7 days.The final dosage was given 2 hours before taking materials and 10%chloral hydrate was injected intraperitoneally.Blood was taken from abdominal aorta in rats and was placed at room temperature for 2 h.The serum of the same group was collected and inactivated at 56℃for 30 minutes.RAW264.7 were induced to differentiate into osteoclasts by different concentrations(25,50,100 ng·ml-1)RANKL for 3,5and 7 days.The positive cells of osteoclasts were observed by TRAP staining technique.The expression of TRACP was evaluated by RT-PCR.Serum containing Zuogui Pills with different concentrations（5,10,15,20%）was screened to intervene in RANKL-induced osteoclasts for 24,48 and 72 hours,MTT assay was used to detect the proliferation of osteoclasts.The positive cells of osteoclasts were observed by TRAP staining technique.The expression of TRACP,RANKL and NFATC1 were evaluated by RT-PCR to observe osteoclastic differentiation.The apoptosis of osteoclasts was observed by AO/EB staining technique and the contents of IL-10 and IL-17 in supernatant were assayed by ELISA.Estrogen receptor（ER）inhibitor ICI 182780 was incubated with 10%serum containing Zuogui Pill for 48 hours,then the number of osteoclasts positive cells was observed by TRAP staining technique.The expression of IL-6,IL-10,IL-10RA,CX3CR1 and CX3CL1 protein was assayed by Western blot.Results:1.Induction and identification of osteoclastsThe results of TRAP staining showed that TRAP-positive cell and nuclei fusion phenomenas could be observed in each group by 5 and 7 d exposure to RANKL.There were a lot of purple-red granules in the cytoplasm,and the area of positive area was proportional to the concentration of RANKL.RT-PCR showed that the expression of TRACP mRNA increased significantly in all groups（P<0.01）when the concentration of RANKL was 50ng·ml-11 and 100 ng·ml-11 for 5 d and was 100 ng·ml-11 for 7 d.2.Effects of serum containing Zuogui Pill on proliferation,differentiation,apoptosis and osteoclast-related inflammatory factors of osteoclastsMTT assay showed that the proliferation of osteoclasts was significantly suppressed after48 hours of intervention with 10%Zuogui Pill medium and high dose serum（P<0.01）.The results of TRAP staining showed that the number of TRAP positive cells decreased in the medium and high dose groups of Zuogui Pill,and most of the cells were regular monocytes.RT-PCR showed that the Zuogui Pill medium dose group could significantly reduce the content of TRACP,RANKL and NFATC1 mRNA in osteoclasts（P<0.01）.The results of AO/EB staining showed that the fluorescence of cells in RANKL group was green and orange-red.Compared with RANKL group,the fluorescence of cells labeled with ethidium bromide in Zuogui Pill medium dose group increased significantly.The results of ELISA showed that the content of IL-10 in the low,medium and high dose groups of Zuogui Pill increased（P<0.05）,and the effect of the medium dose group was more significant（P<0.01）.The content of IL-17 in the medium and high dose groups of Zuogui Pill decreased significantly,and there was no significant difference between them（P>0.05）.3.Serum containing Zuogui Pill regulates osteoclast differentiation and osteoclast-related inflammatory factors through ER Pathway.The results of TRAP staining showed that the number of TRAP positive cells in Zuogui Pill+ICI 182780 group increased after ICI 182780 treatment.Western blot analysis showed that compared with Zuogui Pill group,IL-6 protein expression was significantly increased（P<0.01）,IL-10 protein expression was decreased（P<0.05）,IL-10RA protein expression was increased（P<0.05）,CX3CR1 protein expression was significantly increased in Zuogui Pill+ICI 182780 group（P<0.01）.Conclusion:1.The serum containing Zuogui Pill can inhibit the proliferation and differentiation of osteoclasts induced by RANKL,promote the apoptosis of osteoclasts,effectively increase anti-inflammatory cytokines and reduce pro-inflammatory cytokines.2.The serum containing Zuogui Pill may partly regulate osteoclast-related inflammatory factors through ER pathway to inhibit osteoclastic differentiation and bone resorption function. |
