The Preliminary Research Of PLC-independent PKC Signaling Pathway In PTH Peptide On Bone And Muscle Metabolism

Background:Parathyoid Hormone(PTH)is a commonly used anti-osteoporosis drug in clinically.Previous studies have shown that PTH analogue activates PTH1R to increase cancellous bone mass,improve the microstructure of bone trabecular through nonPLC/PKC signaling pathway.Currently,studies about PTH on bone metabolism are relatively mature,but researches on skeletal muscle metabolism are not enough.Skeletal muscle and bone tissue belong to motor system,play an important role in maintaining the the body's stability and motor function.Both are derived from the mesoderm and express parathyroid hormone receptors.The study of the effects of PTH on bone and muscle metabolism and signaling molecules and pathways will facilitate the design of a better selective signal pathway PTH simulation peptides and inprove the drug efficacy and expand the range of application.Objective:To validate the role of nonPLC/PKC signaling pathway in the regulation of bone metabolism by existing PTH simulation peptide.A specific signal pathway PTH simulation peptide was designed by truncating amino acids and altering the structure of the polypeptide.To explore the role of various signal pathways in PTH regulating metabolism of skeletal muscle.Methods:Thirty-two 7-week old C57BL/6J female mice were used to establish a model of ovariectomized osteoporosis.and PTH simulated peptide was injected subcutaneally.At the fourth weekend,the hindlimb gastrocnemius muscle strength were measured in vivo,and the proximal tibia were subjected to micro-CT cancellous bone quantitative analysis.The proximal tibia was taken for undecalcificated bones slicing for bone morphometric analysis.The paravertebral muscles of the fourth lumbar spine were taken for pathological sectioning.HE staining was performed to observe the muscle fiber area and the number of myofiber satellite cells.The ATPase staining was used to observe the type and quantity of muscle fibers.In addition,BMSC and BMMs were extracted from 8-week-old C57BL/6J mice.Alizarin red staining and calcium quantification,alkaline phosphatase staining,and enzyme activity were detected after osteogenic induction of BMSC.TRAP staining was and enzyme activity assay performed after osteoclast differentiation of BMMs.Extraction of primary osteoblasts from day 3 C57BL/6 mice.Detection of bone metabolism-related gene expression by Real-time PCR.Extraction of gastrocnemius muscle satellite cells from five 4-week-old C57BL/6J mice and then induced by PTH.Detection of muscle metabolism-related gene expression by Real-time PCR.Results:(1)Quantitative cancellous bone:the number of trabecular bone,trabecular thickness,trabecular bone volume fraction in PTH(1-34)group and MY-1 group was significantly higher than model group(P<0.05).There was no statistical difference between the treatment groups and sham operation group.(2)The pathological section of the proximal tibia showed that the percentage of trabecular area in PTH(1-34)group and MY-1 group was significantly higher than that in OVX group(P<0.05).There was no significant difference between SHAM group and bone trabecular mineral deposition rate.The rate of PTH(1-34)group and MY-1 group was significantly higher than that of OVX group(P<0.05),and PTH(1-34)was higher than SHAM group.The number of osteoclasts per unit trabecular bone in the PTH(1-34)group was significantly higher than that in the OVX group(P<0.05).There was no statistical difference between the MY-1 group and the OVX group.(3)Osteogenic differentiation of the BMSC:Alizarin red staining showed that the calcium nodules in the PTH(1-34)group and MY-1 group were significantly higher than those in the control group.Alkaline phosphatase staining showed that dark blue staining in the PTH(1-34)group and the MY-1 group compared with the control group.The ALP quantitatively showed the activity of PTH(1-34)group and MY-1 group was significantly higher than that of the control group(P<0.05).But there was no statistical difference between the PTH(1-34)group and the MY-1 group.(4)Osteoclast differentiation of BMMs:Tartrate-resistant acid phosphatase staining showed that the number of TRAP-positive cells in the PTH(1-34)group was significantly higher than that in the control group and MY-1 group(P<0.05).Quantitative enzyme assays showed that the PTH(1-34)group was significantly higher than the control group and MY-1 group(P<0.05).While there was no statistical difference between the MY-1 group and the control group.(5)Real-time PCR detection of primary osteoblast and BMSC gene expression in the skull:the osteogenic specific genes(ALP,BSP,OC,OPG)expression in PTH(1-34)group and MY-1 group was significantly higher than control group(P<0.05).In the PTH(1-34)group,the expression of Rankl was significantly higher than that in the control group(P<0.05).There was no significant difference between the MY-1 group and the control group.(6)Gastrocnemius muscle strength measurement:The maximal contraction force of OVX group was significantly lower than that of SHAM group(P<0.05).The effective contraction number of OVX group contracting to fibrillation was significantly lower than that of SHAM group(P<0.05).The PTH group was significantly higher than the OVX group.In the OVX group(P<0.05),there was no statistical difference between the MY-1 group and the OVX group.(7)Pathological section of paraspinal muscles:HE staining showed that the average cross-sectional area of muscle fibers in OVX group was larger than that of SHAM,and the average number of single-muscle fiber was decreased(P<0.05).There was no statistical difference between PTH(1-34)group and SHAM group.There was no significant difference between MY-1 group and OVX group.ATPase staining showed that the number of type I and type ?a muscle fibers in OVX group was lower than that in SHAM group.Type ?b muscle fibers were higher than SHAM group(P<0.05),while PTH group I and ?a The number of muscle fibers was higher than that of OVX,and the number of type ?b muscle fibers was lower than that of OVX(P<0.05).(8)Myogenesis-related gene expression:Myogenic myogenic(Myf5,Myf6)of PTH(1-34)group and MY-1 group were significantly higher than con group(MyHcI,MyoG)gene MY-1 group was significantly higher than in the con group(P<0.05),the MyoD gene PTH(1-34)was significantly lower than that of the con group(P<0.05).Conclusion:There is no evidence that the non-PLC/PKC pathway PTH mimetic peptide MY-1 promotes the formation and differentiation of osteoclasts,but can alleviate osteoporosis due to estrogen loss,which is close to that of PTH(1-34).PTH(1-34)can reverse the changes in the structure of myofibers caused by ovariectormy and decrease the contractile force.The non-PLC/PKC signaling pathway mimic peptide MY-1 has a different role than PTH(1-34).