Sirtuin 1 participates in intervertebral disc degeneration via the nicotinamide phosphoribosyl transferase/nicotinamide adenine dinucleotide/sirtuin 1 pathway responsible for regulating autophagy of nucleus pulposus cells

Abstract

Disc degeneration is the main cause of discogenic low back pain, disc herniation, degenerative stenosis of spinal canal, lumbar spondylolisthesis and other diseases. In the process of intervertebral disc degeneration, water and extracellular matrix of nucleus pulposus tissues are lost, so the normal tension in the intervertebral disc cannot be maintained, which worsens the living environment of nucleus pulposus cells. Low back pain (LBP), with a high incidence rate of disability, has become an increasing health concern and a social and economic problem. The present study aimed to analyze the action mechanisms of nicotinamide phosphoribosyl transferase (Nampt) and sirtuin 1 (SIRT1) in intervertebral disc degeneration (IVDD). In total 26 patients with lumbar disc herniation who had surgical resection at The Third Affiliated Hospital of Jinzhou Medical University were recruited as the experimental group and their degenerative nucleus pulposus (DNP) tissues of intervertebral disc were collected. In addition, nucleus pulposus tissues of intervertebral disc were collected from 20 patients with burst fracture of lumbar spine at the same hospital (control). Nucleus pulposus cells from primary culturing were separated for subsequent experimentation. LC3 II/I, beclin-1, SIRT1 and NAMPT mRNA and protein expression levels were determined using reverse transcription-quantitative PCR and western blotting, respectively. Nicotinamide adenine dinucleotide (NAD) contents in nucleus pulposus cells was determined by NAD assay kit. The mRNA and protein expression levels of SIRT1 in DNP tissues were reduced compared with the control tissues and decreased with increasing disease severity. The expression of autophagy-associated LC3 II/I and beclin-1 in DNP tissues was reduced compared with control tissues. SIRT1 regulated the LC3 II/I and beclin-1 expression levels in nucleus pulposus cells. Treatment with resveratrol and inhibitor of SIRT1 showed that Nampt/NAD⁺/SIRT1 pathway participated in the process of IVDD by regulating autophagy of nucleus pulposus cells. SIRT1 serves a role in the process of IVDD through Nampt/NAD⁺/SIRT1 pathway that regulates autophagy of nucleus pulposus cells. SIRT1 may become a biological target for the treatment of IVDD.

Keywords: LC3; beclin-1s; intervertebral disc degeneration; nicotinamide phosphoribosyl transferase; nucleus pulposus; sirtuin 1.

Figure 1

Expression of SIRT1 in nucleus pulposus tissues. (A) SIRT1 mRNA and (B) protein expression levels in control (normal nucleus pulposus) and DNP tissues. ^*P<0.05 compared to control. (C) mRNA expression levels and (D) protein expression levels of SIRT1 in DNP tissues from IVDD patients at grades I, II and III and IVDD patients at grades IV and V. ^**P<0.01 compared with IVDD patients at grades I, II and III. RT-qPCR was used to measure mRNA levels and western blotting was used to assess protein expression. SIRT1, sirtuin 1; RT-q, reverse transcription-quantitative; DNP, degenerative nucleus pulposus IVDD, intervertebral disc degeneration.

Figure 2

Expression of autophagy-associated genes in nucleus pulposus tissues. (A) LC3 and (B) beclin-1 mRNA expression levels in DNP tissues compared with control (normal nucleus pulposus) tissues. RT-qPCR was performed to determine the relative mRNA expression levels. ^*P<0.05 and ^**P<0.01 compared to control. (C) LC3 and (D) beclin-1 protein expression levels in the DNP tissues compared with control (normal nucleus pulposus) tissues. Western blotting was performed to measure the relative protein expression levels. ^*P<0.05 compared to control. RT-q, reverse transcription-quantitative; DNP, degenerative nucleus pulposus.

Figure 3

Effect of SIRT1 on the expression of autophagy-associated genes in nucleus pulposus cells. SIRT1 (A) mRNA and (B) protein expression levels in cells treated with resveratrol or nicotinamide. LC3 (C) mRNA and (D) protein expression levels in cells treated with resveratrol or nicotinamide. Beclin 1 (E) mRNA and (F) protein expression levels in cells treated with resveratrol or nicotinamide. ^*P<0.05 and ^**P<0.01. RT-qPCR was used to measure mRNA levels and western blotting was used to assess protein expression. Resveratrol; SIRT1 agonist; nicotinamide; SIRT1 inhibitor; SIRT1, sirtuin 1; RT-q, reverse transcription-quantitative; control, cells treated with the solvent of the SIRT1 inhibitor.

Figure 4

Nampt/NAD⁺/SIRT1 pathway in nucleus pulposus cells. Nampt (A) mRNA and (B) protein expression levels in control cells and cells treated with FK866. (C) mRNA expression levels and (D) protein expression levels of SIRT1 in control cells and cells treated with FK866. (E) NAD⁺ contents in control cells and cells treated with FK866 (F) LC3II/I and beclin-1 protein expression levels in in control cells and cells treated with FK866. ^*P<0.05 and ^**P<0.01 compared with the control group. RT-qPCR was used to measure mRNA levels and western blotting was used to assess protein expression and NAD⁺ contents were detected with the NAD⁺ concentration determination kit. FK866, Nampt inhibitor; control group, untreated cells; Nampt, nicotinamide phosphoribosyl transferase; NAD, nicotinamide adenine dinucleotide; SIRT1, sirtuin 1; RT-q, reverse transcription-quantitative.