关键词: 枸杞多糖, 椎间盘退变, PI3K/Akt/mTOR, 髓核细胞, 代谢平衡

Abstract: Objective To investigate the regulatory effect of Lycium barbarum polysaccharide(LBP)on the anabolic and catabolic balance of nucleus pulposus(NP)cells during intervertebral disc degeneration(IVDD),and to elucidate its underlying mechanism.Methods In the in vivo experiment,a rat lumbar IVDD model was established by surgical puncture using SD rats.The rats were randomly divided into sham operation group,model group,and low-,medium-,and high-dose LBP groups.After intervention,morphological changes of intervertebral disc tissues were observed by Safranin O-fast green staining and hematoxylin-eosin(HE)staining.NP cell apoptosis was detected by TUNEL staining.The protein expression levels of anabolic markers(typeⅡcollagen,COL2;aggrecan,ACAN)and catabolic markers(matrix metalloproteinase-13,MMP-13;a disintegrin and metalloproteinase with thrombospondin motifs 5,ADAMTS-5)in NP tissues were detected by Western blotting.In the in vitro experiment,Rat NP cells were isolated and cultured,and an IVDD cell model was established using interleukin-1β(IL-1β).The cells were divided into control group,model group,LBP group,and LBP+PI3K inhibitor group.Cell proliferation and apoptosis were assessed by CCK-8 assay and flow cytometry,respectively.The mRNA levels of anabolic markers(COL2,ACAN)and catabolic markers(MMP-13,ADAMTS-5)were detected by qPCR.The phosphorylation levels of key proteins of the PI3K/Akt/mTOR pathway(p-PI3K,p-Akt,p-mTOR)were measured by Western blotting.Results Compared with the model group,each LBP treatment group(especially the medium-and high-dose groups)exhibited significantly alleviated intervertebral disc degeneration in vivo.Intact NP structure and increased Safranin O-fast green staining intensity were observed in histological staining,indicating reduced proteoglycan loss.A marked decrease in NP cell apoptosis rate was revealed by TUNEL staining.After LBP treatment,the expression of COL2 and ACAN were up-regulated in a dose-dependent manner,while the expression of MMP-13 and ADAMTS-5 were down-regulated(all P<0.05).The viability of IL-1β-induced degenerative NP cells was significantly enhanced and cell apoptosis was effectively inhibited in vitro.The phosphorylation of key proteins of the PI3K/Akt/mTOR pathway(p-PI3K,p-Akt,p-mTOR)was promoted,and a metabolic balance-regulating effect was exhibited by promoting COL2 and ACAN expression and inhibiting MMP-13 and ADAMTS-5 expression.These protective effects of LBP were significantly blocked by the addition of a PI3K inhibitor(all P<0.05).Conclusion LBP effectively delays the progression of intervertebral disc degeneration both in vivo and in vitro.Its mechanism may involve activation of the PI3K/Akt/mTOR signaling pathway,thereby positively regulating the anabolic and catabolic balance of nucleus pulposus cells.

Key words: Lycium barbarum polysaccharide, intervertebral disc degeneration, PI3K/Akt/mTOR, nucleus pulposus, metabolic balance