Effect of Serum Containing Zuogui Pill on the Expression of Ihh,Ptch,Smo,GLi1 Proteins in Osteoblasts and the Number of Osteoclasts
Objective Explore the effect of serum containing Zuogui pill on the expression of key proteins in the Hedgehog-GLi pathway of osteoblasts and the number of osteoclasts.Methods 50 female SD rats were randomly divided into control group,sham-operated group,and model group.The model group rats underwent bilateral ovariectomy.After 12 weeks,the rats in the model group were randomly divided into the ovariectomy group(OVX),positive control group,and Zuogui pill group.The positive control group rats were orally administered estradiol valerate suspension,the Zuogui pill group rats were administered with Zuogui pill water solution for 7 days.1.5 hours after the final administration,blood was collected to prepare corresponding drug containing serum.The co-culture system for osteoblasts,osteoclasts,and bone slices was established and divided into blank control serum group,sham-operated serum group,OVX serum group,positive control serum group,Zuogui pill containing serum group,agonist serum group,and agonist+Zuogui pill containing serum group.Corresponding serum was added to each group separately.The protein expression of Ihh,Ptch,Smo,and GLi1 in osteoblasts was detected by immunofluorescence.The protein expression of OPG and RANKL in osteoblasts was detected by immunohistochemistry.The number of TRAP-positive cells was calculated.ELISA was used to detect the content of TRAP in the supernatant of the co-culture system.Results Compared with sham-operated serum group,the protein expression level of Ihh,Ptch,Smo,and GLi1 were significantly upregulated(P<0.01),and the RANKL/OPG ratio increased,while the number of TRAP+cells and the TRAP content in the cellular supernatant also significantly increased in the OVX serum group(P<0.05).Compared with the OVX serum group,the protein expression of Ihh,Ptch,Smo,and GLi1 was significantly down-regulated and the RANKL/OPG ratio decreased,the number of TRAP+cells and the TRAP content in the cell supernatant also significantly decreased(P<0.05)in the Zuogui pill containing serum group.Compared with the agonist serum group,after the addition of Zuogui pill containing serum,the protein expression of Ihh,Ptch,Smo,GLi1 and the RANKL/OPG ratio were significantly down-regulated,and the number of TRAP+cells and TRAP content also significantly decreased(P<0.05).Conclusion By inhibiting the abnormally active Hedgehog-GLi signaling pathway,Zuogui pill containing serum can downregulate the protein expression level of Ihh,Ptch,Smo,and GLi1,reduce the RANKL/OPG ratio,inhibit the number and activity of osteoclasts,thereby reduce bone loss.
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