Effect of pestle needle therapy on the posterior cervical muscle in a rabbit model of cervical spondylosis

doi:10.19852/j.cnki.jtcm.2025.04.009

Abstract

Abstract:

OBJECTIVE: To investigate the effect of pestle needle therapy (PNT) on the posterior cervical muscle (PCM) in a rabbit model of cervical spondylosis (CS) and explore the underlying mechanisms.

METHODS: Rabbits were divided into control, CS models I and II (CS1 and CS2), electroacupuncture (EA), PNT I and II (PN1 and PN2), activator (AVT), and PNT combined with activator (C-AVT) groups. A long-term neck immobilization technique was used to establish a rabbit model of CS. Following completion of modeling, the EA group received electroacupuncture intervention, whereas the CS1, CS2, and C-AVT groups received PNT intervention. The AVT and C-AVT groups received local 740 Y-P injections into the PCM daily. The inflammatory injury to PCM was evaluated based on pain threshold, morphological changes, and interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α levels. PCM fibrosis was evaluated by measuring the positive area (PA) of collagen fibrils (CFs) and collagen type 1 alpha 1 (Col1α1) using Masson's and immunohistochemical staining. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and transmission electron microscopy were used to identify apoptotic cells and assess autophagy, respectively. Western blotting was used to determine B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cysteine aspartate-specific protease (caspase)-3, sequestosome-1 (P62), microtubule-associated protein light chain 3 (LC3-I/II), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) levels. Real-time quantitative polymerase chain reaction was used to determine mRNA expression levels of PI3K, AKT, mTOR, autophagy protein (ATG), and ATG7.

RESULTS: PNT alleviated PCM cell degeneration and necrosis, inhibited inflammatory cell infiltration, decreased IL-1β, IL-6, and TNF-α levels, and decreased the PA of CFs and Col1α1. In the PN1 group, cell apoptosis in the PCM decreased, autophagy increased, Bcl-2 and LC3-II/I levels increased, Bax, Caspase-3, and P62 levels decreased, and the mRNA expression of ATG5 and ATG7 increased. PNT inhibits protein and mRNA expression of PI3K, AKT, and mTOR. Finally, the trend in the results of the rescue experiment was consistent with previous results.

CONCLUSION: PNT inhibited apoptosis and promoted autophagy of PCM cells in CS rabbits and alleviated inflammation and fibrosis injury of PCM by inhibiting the PI3K/AKT/mTOR pathway.

Key words: spondylosis, pestle needle, phosphatidylinositol 3-kinase, proto-oncogene proteins c-akt, TOR serine-threonine kinases, apoptosis, autophagy

WU Haoyang, Liu Yuan, CHEN Yuzhou, XIE Yizhou, ZHONG Lei, YU Yang, FAN Xiaohong. Effect of pestle needle therapy on the posterior cervical muscle in a rabbit model of cervical spondylosis[J]. Journal of Traditional Chinese Medicine, 2025, 45(4): 786-795.

Figures/Tables 4

Figure 1 PNT exerts a positive effect on PCM injury in CS rabbit A: pain threshold; B: HE staining (× 200, scale bar: 100 μm). Yellow arrow: degenerated and necrotic cells; Red arrow: inflammatory cells; Green arrow: fibrous hyperplasia. C: Masson staining (× 400, scale bar: 50 μm); D: calculation of Masson staining PA %; E: IHC staining of Col1α1 (× 200, scale bar: 40 μm). B1, C1, E1: CON; B2, C2, E2: CS1; B3, C3, E3: EA; B4, C4, E4: PN1. F: calculation of Col1α1 PA %. CON group: normal healthy rabbits were immobilized without any restriction on the neck for 42 d; CS1 group: rabbits subjected to CS model induce for 42 d without treatment; EA group: CS model rabbits treated with electroacupuncture once 30 min daily for 21 d; PN1 group: CS model rabbits treated with PNT once 35 min daily for 21 d. PNT: pestle needle therapy; HE: hematoxylin-eosin; CON: control group; CS1: cervical spondylosis group 1; EA: electroacupuncture group; PN1: pestle needle thrapy group 1; IHC: immunohistochemistry; Col1α1: collagen type I alpha 1; PA: positive area. Statistical analyses were measured using one-way analysis of variance followed by Fisher’s least significant difference post hoc test, and the data were expressed as the mean ± standard deviation (n = 6 for each group). Compared with the CON group, aP < 0.001, cP < 0.01; compared with the CS group, eP < 0.001, bP < 0.01, dP < 0.05.

Figure 2 PNT promote PCM apoptosis and promote autophagy in CS rabbits A: TUNEL staining (× 400; scale bar: 50 μm). A1-4: merge; A5-8: DAPI; A9-12: TUNEL. A1, A5, A9: CON; A2, A6, A10: CS1; A3, A7, A11: EA; A4, A8, A12: PN1. Red circle: bright band; green circle: dark band; yellow arrow: M line; light blue arrow: Z line; red arrow: mitochondria; dark blue arrow: sarcoplasmic reticulum; purple arrow: autophagosome. B: calculation of TUNEL-positive cells percentage. C: WB bands for Bax, Bcl-2, and Caspase-3; D: relative protein expression of Bax, Bcl-2, and Caspase-3; E: TEM observation (× 30 000, scale bar: 500 nm). E1: CON; E2: CS1; E3: EA; E4: PN1. F: WB bands for P62, LC3-I, and LC3-II. G: relative protein expression of P62, and LC3-II/I. H: relative mRNA level of ATG5, ATG7. CON group: normal healthy rabbits were immobilized without any restriction on the neck for 42 d; CS1 group: rabbits subjected to CS model induce for 42 d without treatment; EA group: CS model rabbits treated with electroacupuncture once 30 min daily for 21 d; PN1 group: CS model rabbits treated with PNT once 35 min daily for 21 d. TUNEL: terminal deoxynucleotidyl transferase-mediated DUTP nick end labeling; DAPI: 4',6-Diamidino-2-phenylindole; CON: control group; CS1: cervical spondylosis group 1; EA: electroacupuncture group; PN1: pestle needle therapy group 1; WB: Western bolt; Bax: Bcl-2 associated X protein; Bcl-2: B-cell lymphoma-2; Caspase-3: cysteine aspartate-specific protease-3; TEM: transmission electron microscope; P62: sequestosome-1; LC3: microtubule-associated protein light chain 3; ATG: autophagy protein; mRNA: messenger ribonucleic acid; PNT: pestle needle therapy; CON: control; CS: cervical spondylosis; EA: electroacupuncture; PN: pestle needle. Statistical analyses were measured using one-way analysis of variance followed by Fisher’s least significant difference post hoc test, and the data were expressed as the mean ± standard deviation (n = 6 for each group). Compared with the CON group, aP < 0.001, eP < 0.01; compared with the CS group, cP < 0.001, bP < 0.01, and dP < 0.05.

Figure 3 PNT inhibit the PI3K/AKT/mTOR pathway in CS rabbits A: WB bands for PI3K, AKT, and mTOR; B: relative protein expression of PI3K, AKT, and mTOR; C: relative mRNA level of PI3K, AKT, and mTOR. CON group: normal healthy rabbits were immobilized without any restriction on the neck for 42 d; CS1 group: rabbits subjected to CS model induce for 42 d without treatment; EA group: CS model rabbits treated with electroacupuncture once 30 min daily for 21 d; PN1 group: CS model rabbits treated with PNT once 35 min daily for 21 d. CON: control group; CS1: cervical spondylosis group 1; EA: electroacupuncture group; PN1: pestle needle therapy group 1; WB: Western bolt; mRNA: messenger ribonucleic acid; PI3K: phosphatidylinositol 3-kinase; AKT: protein kinase B; mTOR: mammalian target of rapamycin; PNT: pestle needle therapy; CON: control; CS: cervical spondylosis; EA: electroacupuncture; PN: pestle needle. Statistical analyses were measured using one-way analysis of variance followed by Fisher’s least significant difference post hoc test, and the data were expressed as the mean ± standard deviation (n = 6 for each group). Compared with the CON group, aP < 0.001, dP < 0.01; compared with the CS group, bP < 0.01, dP < 0.05.

Figure 4 Rescue experiment A: WB bands for PI3K, P62, LC3-I, LC3-Ⅱ, Bax, Bcl-2 and Caspase-3 staining in PCM tissues; B: relative protein expression of PI3K, P62, and LC3-Ⅱ/Ⅰ; C: relative protein expression of Bax, Bcl-2 and Caspase-3. AVT group: CS model inducing by LNI for 42 d and received a 2 mL 740 Y-P (0.2 g/L) injection in the PCM for 5 d; C-AVT group: CS model rabbits treated with PNT for 21 d and received a 2 mL 740 Y-P (0.2 g/L) injection in the PCM for 5 d; CS2 group: CS model inducing by LNI for 42 d without treatment; PN2 group: CS model rabbits treated with PNT once 35 min daily for 21 d. AVT: activators group; C-AVT: pestle needle combined activators group; CS2: cervical spondylosis group 2; PN2: pestle needle therapy group 2; LNI: long-term neck immobilization; WB: Western bolt; PI3K: phosphatidylinositol 3-kinase; P62: sequestosome-1; LC3: microtubule-associated protein light chain 3; Bax: Bcl-2 associated X protein; Bcl-2: B-cell lymphoma-2; Caspase-3: cysteine aspartate-specific protease-3. The data were expressed as the mean ± standard deviation of the mean (n = 5 for each group). Compared with the C-AVT group, aP < 0.001; compared with the PN2 group, bP < 0.001, eP < 0.01, hP < 0.05; compared with the AVT group cP < 0.001, fP < 0.01, iP < 0.05; compared with the CS2 group, dP < 0.001, gP < 0.01.

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