Jingui Shenqi Pills Regulates Bone-fat Balance In Murine Postmenopausal Osteoporosis With Kidney Yang Deficiency

Objective(1)To construct a model of postmenopausal osteoporosis(PMOP)with kidney-yang deficiency in mice;(2)To prove that Jingui shenqi pills(JGSQP),a classic traditional Chinese medicine formula for warming and tonifying kidney-yang,improves bone-fat balance to treat PMOP with kidney-yang deficiency syndrome in mice.Methods(1)Establishment of animal model:To construct a model of PMOP with kidney-yang deficiency in mice,14 six-week-old C57/BL6 mice were bred adaptively for 2 weeks and randomly divided into two groups:Sham group(n=8)and Ovariectomy(OVX)group(n=8).The syndrome characteristics of kidney-yang deficiency in mice were observed,and the quantitative indexes of kidney-yang deficiency such as rectal temperature,water and food intake,and wet weight of padding were examined.Eight weeks later,the mice were drawn sampling,and micro-CT examination of L4 vertebrae bone microstructure confirmed the establishment of PMOP model in mice.(2)Treatment experiment:To confirm whether JGSQP can treat PMOP with kidney-yang deficiency syndrome by improving bone-fat balance,7-week-old female C57BL/6 mice were adaptively reared for one week and randomly divided into three groups: Sham operation group(removal of fat around the bilateral ovaries);Model group(PMOP with kidney-yang deficiency syndrome in mice): JGSQP group(after the establishment of the PMOP with kidney-yang deficiency syndrome model in mice,JGSQP treated the mice for 4 weeks).The mice in the sham operation group and the model group were intragastrically administered with the same amount of normal saline for 4 weeks.The three groups of mice were all euthanized at 12 weeks and samples were taken.The syndrome characteristics of kidney-yang deficiency in mice were observed,and the quantitative indexes of kidney-yang deficiency such as rectal temperature,water intake,food intake,and wet weight of padding were examined.Micro-CT was used to detect the microstructure of vertebral bone.H&E staining was used to detect bone histomorphology,and the number and proportion of vertebral osteoblasts and adipocytes were quantitatively analyzed.q PCR was employed to calculate the expression of vertebral Akt and osteogenic genes(Wnt10b,Osterix),and adipogenic genes(Fndc5,Pparγand Fabp4).Western blot was employed to detect Akt,phosphorylated-Akt(p-AKT),and osteogenic proteins(BMP2 and COL1A1),and adipogenic proteins(Fndc5).Results(1)Construct a model of PMOP with kidney-yang deficiency in mice:①Evaluation of PMOP with kidney-yang deficiency syndrome in mice: 8 weeks after ovariectomy,the mice showed at least three symptoms or signs: loss of luster in hair or sparse and upright hair,listlessness,unwillingness to move,slow response,less food intake or water drinking.After quantitative evaluation of the relevant symptoms and signs,it was found that the rectal temperature decreased(P<0.05)and the wet weight of padding increased(P<0.05)in OVX group compared to Sham group.There is a downward trend in the amount of water and food intake in OVX group.②Lumbar Micro-CT analysis: The three-dimensional reconstruction images showed that the loss of cancellous trabecular bone mass in the vertebrae at eight weeks postoperatively was significant,the bone microstructure was destroyed,and the trabecular spacing was expanded in the OVX group.The quantitative analysis of the three-dimensional reconstruction images showed that bone volume fraction(BV/TV),number of trabecular bone(Tb.N),trabecular bone thickness(Tb.Th)decreased,trabecular separation(Tb.Sp)increased in the OVX group compared with the sham operation group(all P<0.05).(2)JGSQP improves bone-fat balance to treat PMOP with kidney-yang deficiency syndrome in mice:①Evaluation of kidney-yang deficiency syndrome of PMOP in mice: JGSQP could improve the kidney-yang deficiency syndrome of mice,increase the rectal temperature of mice(P<0.05),and decrease the wet weight of padding(P<0.05).The amount of water and food intake tended to increase in JGSQP group.②Micro-CT analysis of lumbar spine in mice: After the intervention of JGSQP,the trabecular bone thickened,the trabecular bone spacing became smaller,and BV/TV,Tb.N and Tb.Th all increased(P<0.05),the ratio of bone surface area to tissue volume(BS/TV)had a tendency to increase,and Tb.Sp and structural model index(SMI)had a tendency to decrease.③H&E staining analysis of lumbar spine in mice: After the intervention of JGSQP,the bone trabecula increased and the fat vacuole decreased.Quantitative analysis of bone tissue morphology shows that JGSQP could increase the number of osteoblasts(Ob.N/BS)and the area of osteoblasts(Ob.S/BS),and reduce the number of adipocytes(adipocytes/tissue area)and adipocytes area/tissue area(P<0.05).④qPCR analysis of lumbar vertebral bone in mice: After the intervention of JGSQP,Akt expression showed a downward trend,bone formation-related gene(Wnt10b)increased(P<0.05),Osx expression showed an increasing trend,adipogenesis-related gene(Fndc5)decreased(P<0.05),and Pparγ,Fabp4 has a downward trend.⑤Analysis of protein expressions in the lumbar spine in mice: After treatment with JGSQP,the expression of p-AKT protein increased(P<0.05),the expression of osteogenicrelated protein(BMP2)increased(P<0.05),and COL1A1 protein showed an increasing trend,adipogenesis-related protein(FNDC5)decreased(P<0.05).Conclusion(1)Establishment of animal model: C57 mice(8 weeks old)can establish a model of PMOP with kidney-yang deficiency in mice after 8 weeks of ovariectomy.(2)Therapeutic experiment: JGSQP may regulate the bone-fat balance to treat PMOP with kidney-yang deficiency syndrome in mice through AKT pathway.