Tuina Alleviates Neuropathic Pain in CCI Rats by Regulating the TRPV4-CaMKII Signaling Pathway in Dorsal Root Ganglion

Abstract

Background: Peripheral sensitization mediated by the Transient Receptor Potential Vanilloid 4-Calcium/calmodulin-dependent protein kinase II (TRPV4-CaMKII) signaling pathway plays a fundamental role in the generation and maintenance of neuropathic pain (NP). Tuina, a safe and effective therapy in traditional Chinese medicine, shows analgesic effects; however, the underlying mechanisms remain unclear. We aimed to investigate whether Tuina alleviates pain by modulating the TRPV4-CaMKII/CREB/NLRP3 signaling pathway. Methods: The Chronic Constriction Injury (CCI) model of the sciatic nerve was used to simulate clinical NP. Tuina was applied to the Yinmen (BL37), Yanglingquan (GB34), and Chengshan (BL57) acupoints once daily for 14 days. Mechanical Withdrawal Threshold (MWT) and Thermal Withdrawal Latency (TWL) were assessed to evaluate the analgesic effect of Tuina. Its protective effects on dorsal root ganglion (DRG) neurons were evaluated using Nissl staining. The whole-cell patch clamp technique recorded excitability changes in DRG neurons and assess the effects of Tuina on peripheral sensitization. Western blot (WB), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) helped detect changes in the TRPV4-CaMKII/CREB/NLRP3 pathway and expression of inflammation-related cytokines in DRG neurons. Results: Tuina significantly alleviated mechanical allodynia and thermal hyperalgesia in CCI rats and exerted a protective effect on DRG neurons. Patch clamp recordings showed that Tuina inhibited hyperexcitability in DRG neurons. Mechanistically, Tuina downregulated the expression of the TRPV4-CaMKII/CREB/NLRP3 signaling pathway and reduced the secretion of TNF-α, IL-1β, and IL-18. Conclusion: The analgesic effect of Tuina in CCI rats is associated with reduced peripheral sensitization via modulation of the TRPV4-calcium signaling cascade.

Keywords: TRPV4; Tuina; dorsal root ganglion; neuropathic pain; peripheral sensitization.

Figure 1

Experimental timeline and Tuina acupoints. (a) Timeline of the experiment and (b and c) the relative positions of the Yanglingquan (GB34), Yinmen (BL37), and Chengshan (BL57) acupoints and their corresponding acupoints in humans.

Figure 2

Effect of Tuina on CCI-induced mechanical allodynia and thermal hyperalgesia (n = 6). (a) The MWT results for the injured hindpaws of rats. (b) The TWL results for the injured hindpaws of rats. ^##p < 0.01 vs. Sham. ^∗∗p < 0.01 vs. CCI.

Figure 3

Effect of Tuina on Nissl bodies in DRG neurons of CCI rats (n = 3). (a–c) Representative images of Nissl staining in the DRG. Original magnification, ×  400 and (d) the number of Nissl body in the DRG. ^∗∗p < 0.01.

Figure 4

The effect of Tuina on TRPV4 ion channels in the DRG of CCI rats (n = 3). (a) Representative images of immunofluorescence of TRPV4 in DRG; (b) immunofluorescence quantification of TRPV4; and (c and d) representative gels and quantification of TRPV4. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 5

Results of behavioral tests in each group of rats (n = 8). (a) Results of MWT in rats and (b) results of TWL in rats. ^##p < 0.01 vs. Sham. ^∗∗p < 0.01.

Figure 6

The impact of Tuina on electrophysiological alterations in DRG neurons of CCI rats (n = 6-7 cells). (a) Illustration of action potentials in DRG neurons; (b) number of action potentials of neuronal cells in DRG neurons; (c) changes in the resting membrane potential in DRG neurons. ^∗∗p < 0.01.

Figure 7

Effects of Tuina on CaMKII/CREB/NLRP3 pathway and proinflammatory cytokines TNF-α, IL-1β, and IL-18 in CCI Rats (n = 3). (a) Representative images and quantitative analysis of immunofluorescence of p-CaMKII; (b) representative Western blot bands of P-CaMKII, CaMKII,P-CREB, CREB, and NLRP3 in the each group; (c) quantitative analysis of P-CaMKII/CaMKII, P-CREB/CREB, and NLRP3/β-actin observed in Western blots; and (d–f) secretion of TNF-α, IL-1β, and IL-18 as detected by ELISA. ^∗p < 0.05, ^∗∗p < 0.01.