Experimental Study Of Naringin Protects Human Degenerative Nucleus Pulposus Cells Against Senescence Via Autophagy

Objective:To observe the autophagy level and the viability of human degenerative nucleus pulposus(NP)cells with different doses of naringin and autophagy inhibitor in-vitro.Accordingly,we explored the autophagy activity of human NP cells and correlation between them in senescence and metabolic level.Based on the basic theory of Traditional Chinese Medicine(TCM)of “kidney-invigorating and blood-activating”,we investigated whether naringin could protect against human NP cell senescence,metabolic imbalance,abnormal secretion of catabolic enzymes and extracellular matrix degradation via autophagy.Thus,it discovered the potential effect of naringin and provided a new research direction for TCM in treating intervertebral disc degeneration.Methods:According to exclusion criteria and inclusion criteria,human NP samples were acquired from 10 patients with a diagnosis of lumbar disc herniation who were undergoing surgery in our hospital from February 2021 to December 2021(type of kidney deficiency and blood stagnation in TCM).Human NP cells were isolated and cultured in-vitro.Identification of human NP cell marker Collagen Ⅱ by Collagen Ⅱ immunocytochemical staining.The optimal dose of naringin for human NP cell was evaluated by CCK8 assay.All human NP cells were divided into four groups(including lowdose naringin group,medium-dose naringin group,high-dose naringin group and autophagy inhibitor group)according to arithmetic progression.Human NP cells were taken to track on the efficiency of autophagy with p AV-CMVTag RFP-SEP-LC3 recombinant adenovirus vectors.We observed the ultrastructure of the autophagosomes under a transmission electron microscopy(TEM).The state of human NP cell senescence was detected by senescence β-galactosidase staining.The activity of human NP cell metabolism was evaluated by ATP colorimetric assay.The micro-RNA expression of MMP-3 and Collagen Ⅱ were analyzed using quantitative realtime PCR.The target protein(LC3,Beclin-1,P62,MMP-3 and CollagenⅡ)expression was measured by Western blot assay.Results: 1.Collagen Ⅱ immunocytochemical staining was positive to verify the human NP cells.2.In the CCK8 assay,10 μg/m L naringin treatment showed the strongest protective effects in NP cells.All collected NP cells were divided into four groups,which including low-dose naringin group(5 μg/m L),medium-dose naringin group(10 μg/m L),high-dose naringin group(15 μg/m L)and autophagy inhibitor group(10 μM 3-MA).3.We observed more autophagosomes in the group of low-dose naringin,medium-dose naringin and high-dose naringin versus autophagy inhibitor group under TEM.4.The results of p AVCMV-Tag RFP-SEP-LC3 assay showed that medium-dose naringin group significantly increased the number of green fluorescence intensity spots(p﹤0.05)versus low-dose naringin group and autophagy inhibitor group markedly decreased(p﹤0.001),but there was no significant difference in the high-dose naringin group(p﹥0.05).5.Medium-dose naringin group significantly increased the ratio of LC3Ⅱ/Ⅰ versus low-dose naringin group(p﹤0.05),but autophagy inhibitor group markedly inhibited the ratio of LC3Ⅱ/Ⅰ and the expression of Beclin-1(p﹤0.05,p﹤0.01).There was no significant difference among other groups(p ﹥ 0.05).6.Autophagy inhibitor group significantly enhanced the expression of P62 versus low-dose naringin group,but there was no significant difference among other groups(p﹥0.05).7.The results of β-galactosidase staining revealed that autophagy inhibitor treatment significantly accelerated the process of NP cell senescence versus lowdose naringin treatment(p﹤0.05).8.The assay of ATP colorimetric showed that medium-dose naringin treatment markedly increased ATP generation versus low-dose naringin treatment(p﹤0.05),but autophagy inhibitor treatment significantly decreased the synthesis of ATP(p﹤0.05).9.The results of quantitative real-Time PCR showed that medium-dose naringin group significantly enhanced the micro-RNA expression of Collagen Ⅱversus low-dose naringin treatment(p﹤0.05),but autophagy inhibitor group markedly increased the micro-RNA expression of MMP-3 and decreased the micro-RNA expression of Collagen Ⅱ(all p﹤0.01).10.The results of Western blot assay revealed that medium-dose naringin treatment markedly increased the synthesis of Collagen Ⅱ and decreased the expression of MMP-3 versus low-dose naringin treatment(p ﹤ 0.001,p ﹤ 0.05),but autophagy inhibitor treatment significantly decreased the expression of Collagen Ⅱ and promoted the expression of MMP-3(p﹤0.01,p﹤0.05).11.The optimum concentration of naringin was 10 μg/m L.Conclusion:1.Naringin treatment enhanced the expression of autophagy and increased autophagic flux.2.The autophagy activity of human NP cell was boosted by naringin,which ameliorated human NP cell senescence,metabolism,decomposition enzyme activity and the extracellular matrix components.