维生素D在椎间盘退变中的作用及机制研究OA

Objective:To investigate the effects of Vitamin D on intervertebral disc degeneration(IDD)in rats and to eluci-date its protective mechanism.Methods:An acupuncture-induced IDD model was established.Rats were randomly divided into four groups(n=10 per group):sham-operated group,model group,Vitamin D(0.15 μg/kg)group,and Vitamin D(0.15 μg/kg)+AMP-activated protein kinase(AMPK)inhibitor(Compound C,20 mg/kg)group.One week after mod-el establishment,the corresponding drugs were administered via gavage/intraperitoneal injection once daily for 4 consecu-tive weeks.The degree of disc degeneration was assessed using the disc height index(DHI).Pathological changes in the caudal intervertebral discs were observed by Hematoxylin-Eosin(HE)and Safranin O-Fast Green staining.The level of apoptosis in nucleus pulposus(NP)tissue was detected by TUNEL assay.Immunohistochemical staining was performed to measure the expression levels of Aggrecan,matrix metalloprotein-ase 13(MMP-13),Cleaved Caspase-3,B-cell lymphoma 2(Bcl-2),and Bcl-2-associated X protein(Bax)in NP tissue.Mitochondrial structural changes in NP cells were observed using transmission electron microscopy(TEM).Biochemical assays were used to determine the adenosine triphosphate(ATP)content and the activity levels of mitochondrial respira-tory chain complexes Ⅰ and Ⅱ in NP tissue.Western Blot was employed to detect the expression of Mitofusin 1(Mfn1),Mitofusin 2(Mfn2),Peroxisome proliferator-activated receptor γ coactivator 1α(PGC-1α),Dynamin-related protein 1(Drp1),AMPK,and its phosphorylated form(p-AMPK).Results:Compared with the sham-operated group,the model group exhibited a significantly lower DHI(P<0.05),disorganized disc structure,atrophy of the nucleus pulposus,rupture of the annulus fibrosus,and loss of cartilaginous endplate matrix,indicating significant degenerative changes.In the NP tis-sue,the levels of MMP-13,apoptosis rate,Cleaved Caspase-3,Bax,and the p-Drp1/Drp1 ratio were significantly increased(P<0.05),while the levels of Aggrecan,Bcl-2,ATP,respiratory chain complexes Ⅰ/Ⅱ,Mfn1,Mfn2,p-AMPK/AMPK ratio,and PGC-1α were significantly decreased(P<0.05).Severe mitochondrial structural damage was also observed.Compared with the model group,the Vitamin D group showed a significantly higher DHI(P<0.05),a significant mitiga-tion of disc degeneration was observed,characterized by a structural normalization with exhibited only slight disorganiza-tion.Concomitantly,the NP tissue showed a marked decrease in several key degenerative indicators,including the levels of MMP-13,apoptotic rate,Cleaved Caspase-3,Bax,and the p-Drp1/Drp1 ratio(P<0.05),while the levels of Aggrecan,Bcl-2,ATP,respiratory chain complexes Ⅰ/Ⅱ,Mfn1,Mfn2,p-AMPK/AMPK ratio,and PGC-1α were significantly increased(P<0.05).Mitochondrial structural damage was also ameliorated.Compound C was found to inhibit the ameliorative effects of Vitamin D on IDD in rats(P<0.05).Conclusion:Vitamin D can delay IDD in rats,restore mitochondrial func-tion,and inhibit apoptosis in nucleus pulposus tissue.Its mechanism of action may be related to the activation of the AMPK/PGC-1α pathway,thereby improving mitochondrial dynamics in nucleus pulposus cells.