Effect of electroacupuncture of "Jiaji"(EX-B2) on autophagy and apoptosis of nucleus pulposus cells in degenerative intervertebral disc rabbits based on PI3K/AKT/mTOR pathway
Objective: To observe the effect of electroacupuncture (EA) of bilateral "Jiaji" (EX-B2) on autophagy, apoptosis and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway of nucleus pulposus cells in rabbits with intervertebral disk degeneration (IVDD), so as to explore its possible mechanisms underlying improvement of IVDD.
Methods: A total of 18 male New Zealand rabbits were randomly and equally divided into sham operation, model and EA groups. The IVDD model was established by axial disc compression, and the modeling effect was confirmed by magnetic resonance imaging (MRI). EA (2 Hz/15 Hz, 1 mA) was applied to bilateral "Jiaji" (EX-B2) of L4-L5 for 20 min, once daily, 6 times a week for 4 weeks. The general condition and body weight change in each group were recorded. The morphology of nucleus pulposus was observed after H.E. staining. The ultrastructure of nucleus pulposus cells was observed by using transmission electron microscope. The apoptosis of nucleus pulposus cells was detected by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The expressions of Bax, Bcl-2, Beclin1, light chain 3 (LC3), autophagy-related protein 5 (Atg5), p62 and PI3K/AKT/mTOR signaling pathway proteins were detected by Western blot.
Results: Compared with the sham operation group, the general condition of IVDD rabbits was poorer, with decreased MRI signal of intervertebral disc, destruction and necrosis of nucleus pulposus cells, damage of nucleus pulposus cell matrix, and blurred organelles, and the apoptosis rate, expression levels of Bax, Beclin1 and Atg5 proteins and the ratio of LC3- Ⅱ/LC3- Ⅰ were significantly increased (P<0.01, P<0.05), while the body weight, expressions of Bcl-2 and p62, p-PI3K/PI3K, p-AKT/AKT and p-mTOR /mTOR considerably decreased (P<0.01) in the model group. In contrast to the model group, the general condition of the rabbits was improved after EA, with enhanced MRI signal of intervertebral disc, fewer mitochondrial autophagosomes, slightly normal mitochondrial construction, etc., and modeling-induced increase of apoptosis rate, the expression levels of Bax, Beclin1 and Atg5 proteins and the ratio of LC3-Ⅱ/LC3- Ⅰ , and decrease of body weight, the expression levels of Bcl-2, p62, and p-PI3K/PI3K, p-AKT/AKT and p-mTOR /mTOR were all reversed in the EA group (P<0.01, P<0.05).
Conclusions: EA stimulation of EX-B2 can delay IVDD by regulating apoptosis and autophagy of nucleus pulposus cells in the intervertebral disc in IVDD rabbits, which may be related to its function in activating PI3K/AKT/mTOR signaling pathway.