| Purpose: Observe the effect of pilose antler polypeptides on autophagy of nucleus pulposus cells of intervertebral discs,explore the mechanism of pilose antler polypeptides protecting intervertebral discs and delaying intervertebral discs degeneration based on Akt-mTOR signaling pathway,and analyze the guiding role of the traditional Chinese medical theory of "Zhi Ze Sheng Hua" in the application of pilose antler polypeptides in rat model of intervertebral disc degeneration.Material and method:60 healthy SD rats with 1 month year old randomly divided into5 groups:normal group,control group,pilose antler polypeptide(PAP)group,rapamycin(Rap)group,pilose antler polypeptide + rapamycin(PAP+Rap)group.Each group has 12 rats.To build models with double forelimb castration method,for control?PAP?Rap and PAP+ Rap groups,then increase water and food feeding height every week.After 8 weeks,the blank group don't do any treatment,the control group with normal saline lavage,PAP group with concentration of 4.5 mg/ml of pilose antler polypeptide lavage,Rap group with concentration of 0.2 mg/ml rapamycin intraperitoneal injection,PAP+Rap group was intraperitoneally injected with rapamycin(0.2mg/ m L)and gavaged with pilose antler polypeptide(4.5mg/ m L).All groups were given the drug for 8 weeks.After administration,half of the samples in the 5 groups collect the L5/6 intervertebral discs and adjacent upper and lower vertebral bodies.After deproteinization,paraffin fixed sections were stained with hematoxylin-eosin staining and Alecine blue staining,and observed under electron microscope.The other half take complete L5/6 disc tissue samples,after liquid nitrogen tank quick-frozen to-80? refrigerator cryopreserved,Western Blot method to detect the expression of p-mTOR,p-AKT related to AKT-mTOR signaling pathways and LC3-??LC3-?related to autophagy.Results:1.Observation by light microscopeUnder hematoxylin-eosin staining,the intervertebral disc annulus aging?the intervertebral disc herniated and the nucleus pulposus shrank was obvious in the control group.Compared with the control group,the intervertebral disc degeneration was significantly reduced in the antler polypeptide group,rapamycin group and antler polypeptide + rapamycin group.The degree of intervertebral disc degeneration was the lowest in the antler polypeptide group,but no difference was observed between the latter two groups.In the blank group,no degenerative signs were observed in disc morphology.Under Alcanblue staining,the contents of proteoglycan and hyaluronic acid in intervertebral disc of PAP?Rap and PAP+ Rap group were significantly increased compared with the control group,and the contents were compared with PAP + Rap group > Rap group > PAP group.No nuclei were seen in each group.2.Western Blot testAkt-mTOR pathway protein detection: p-Akt(phosphorylated Akt)expression level was as follows: PAP + Rap group >PAP group >control group > RAP group(P<0.05);The expression level of p-mTOR(phosphorylated mTOR)was arranged as: PAP + Rap group =PAP group >control group > RAP group,there was no significant difference between the PAP+ Rap group and the PAP group.Rapamycin can inhibit mTOR protein activation,while PAP can promote Akt-mTOR pathway.Autophagy detection: LC3-? expression level is arranged as follows: RAP group > PAP +Rap group > control group > PAP group;LC3-? / ?ratio arrangement as follows: RAP group > PAP + Rap group > control group > PAP group.Rapamycin can increase autophagy level in nucleus pulposus cells,while PAP can decrease autophagy level in nucleus pulposus cells.Conclusion:1.PAP can delay the degeneration of intervertebral disc in rats treated with double forelimb castration.2.PAP can increase the activation of Akt and mTOR in intervertebral disc nucleus pulposus cells,increase the inhibitory effect on downstream autophagy related factors,further reduce the excessive autophagy in nucleus pulposus cells. |
PDF Full Text Request