Study On The Effect Of Serum Containing Zuogui Pill On Bone Resorption Function Of Osteoclasts Based On Hedgehog-Gli Pathway

ObjectiveThe results of previous animal experiments in our group showed that the mRNA and protein expression of Ihh,Smo,Ptch and GLi-1,the key factors of Hedgehog pathway in ovariectomized(OVX)rats,increased significantly,the ratio of RANKL/OPG increased,the activity of OC increased abnormally,and the bone mass decreased.Zuogui Pill can significantly reduce the key factors of Hedgehog pathway,increase bone mineral density and improve the micromorphology of bone trabeculae in OVX rats.Zuogui Pill has obvious therapeutic effect on osteoporosis in OVX rats.On this basis,the co-culture system of osteoblasts,osteoclasts and bone grinding tablets was established to further explore the effect of drug-containing serum of Zuogui Pill on osteoclast bone resorption function from the point of view of Hedgehog-GLi pathway,so as to further reveal the mechanism of Zuogui Pill in the treatment of postmenopausal osteoporosis and provide further experimental basis for TCM treatment of the disease.This study is divided into the following two experimentsMethodsExperimentl Effect of Drug-containing Serum of Zuogui pill on differentiation and function of osteoclastsThe co-culture system of osteoblasts,osteoclasts and bone grinding tablets was established,and the drug-containing serum of rats in each group was prepared.The experimental groups are as follows:①blank control group:Co-culture system+blank control group rat serum②sham operation group:co-culture system+sham operation group rat serum ③OVX group:co-culture system+OVX group rat serum ④positive control group:co-culture system+E group rat serum⑤high dose Zuogui pill group:co-culture system+high-dose Zuogui pill group rat serum(15%drug-containing serum)⑥middle dose Zuogui pill group:co-culture system+medium-dose Zuogui pill group rat serum(10%drug-containing serum)⑦low dose Zuogui pill group:co-culture system.+low-dose Zuogui pill group rat serum(5%drug-containing serum).After 24 hours of co-culture,the cells were replaced by the culture medium of rat serum of each group,and after 7 days of coculture,the cells were collected for follow-up experiment.Osteoclasts were stained with TRAP,the number of TRAP+cells was observed and calculated,and the percentage of bone resorption lacunae was calculated by staining bone resorption lacunae with toluidine blue.Experiment 2.Effect of drug-containing serum of Zuogui pill on Hedgehog-GLi signal PathwayThe experimental groups were as follows:①blank control group:co-culture system+blank control group rat serum②sham operation group:co-culture system+sham operation group rat serum②OVX group:co-culture system+OVX group rat serum④positive control group:co-culture system+E group rat serum⑤Zuogui pill group:co-culture system+ 10%Zuogui pill group rat serum⑥agonist group:coculture system+N-Shh(Hedgehog-GLi signal pathway agonist)+OVX group rat serum ⑦agonist+Zuogui pill group:co-culture system+N-Shh+10%Zuogui pill group rat serum.The differentiated osteoclasts were stained with TRAP,the number of TRAP+cells was observed and calculated,the percentage of bone resorption lacunae was calculated by toluidine blue staining and the morphology of bone lacunae was observed,the content of TRAP in the supernatant of co-culture system was detected by ELISA method,and the mRNA expression of Ihh,Ptch,Smo,GLi1,OPG and RANKL in osteoblasts was detected by Real-timePCR.The protein expression of Ihh,Ptch,Smo and GLil in osteoblasts was detected by cellular immunofluorescence method,and the protein expression of OPG and RANKL was detected by cellular immunohistochemical method.Results(1)The number of TRAP+cells and the area of bone resorption lacunae in the OVX group were significantly higher than those in the sham operation group.Compared with the OVX group,the number of TRAP+cells and the area of bone resorption lacunae in the middle and high dose of Zuogui pills group decreased significantly.Therefore,10%drug-containing serum of Zuogui pill was used for follow-up experiment.(2)The number of TRAP+cells and the area of bone resorption lacunae and the content of TRAP in the supernatant of OVX group were significantly higher than those in sham operation group,while the number of TRAP+cells and the area of bone resorption lacunae in positive control group and Zuogui pill group were significantly lower than those in OVX group,and the content of TRAP in cell supernatant was significantly lower than that in sham operation group.The expression of mRNA and protein of Ihh,Ptch,Smo and GLil in OVX group was significantly higher than that in sham operation group,and compared with sham operation group,the expression of OPG mRNA and protein in osteocytes composed of OVX decreased significantly,the expression level of RANKL mRNA and protein increased significantly,and the ratio of RANKL/OPG increased significantly.After administration of drug serum containing estrogen and Zuogui pills,compared with OVX group,the mRNA and protein expression of Ihh,Ptch,Smo and GLi1 in positive control group and Zuogui pill group were significantly down-regulated,OPG mRNA and protein expression levels were significantly increased,RANKL mRNA and protein expression levels were significantly decreased,and RANKL/OPG ratio was significantly decreased.In OVX group,the expression of mRNA and protein,the key factor of Hedgehog-GLi signaling pathway,was significantly higher than that in OVX group after the addition of agonist Shh recombinant protein.Compared with the agonist group,the mRNA and protein expression and RANKL/OPG ratio of Ihh,Ptch,Smo and GLi1 in the agonist+Zuogui pill group were significantly decreased.There was no statistical difference in the above indexes between the sham operation group and the blank control group,thus excluding the influence of operation factors on the experimental results.ConclusionOVX rat serum can significantly up-regulate the mRNA and protein expression of Ihh,Ptch,Smo and GLi1,the key factors of Hedgehog pathway in osteoblasts,and significantly increase the ratio of RANKL/OPG.At the same time,it can significantly increase the number and activity of osteoclasts,resulting in a significant enhancement of bone resorption function.The drug-containing serum of Zuogui Pill can reverse the above indexes to some extent.Combined with the previous experimental results in vivo,Zuogui Pill can weaken the bone resorption function of osteoclasts by inhibiting the activity of Hedgehog pathway,so as to achieve the effect of treating postmenopausal osteoporosis.