Effects and Mechanisms of Wrist-Ankle Acupuncture on Inflammatory Pain in the Rat Gastrocnemius Muscle [Letter]

Dear editor

We read with great interest the article by Mai et al¹ recently published in the Journal of Pain Research, entitled “Effects and Mechanisms of Wrist‑Ankle Acupuncture on Inflammatory Pain in the Rat Gastrocnemius Muscle”. The authors established a rat model of inflammatory muscle pain induced by CFA injection into the gastrocnemius muscle, and compared the therapeutic effects of traditional WAA‑AS versus WAA‑TENS using transcriptomic analysis. This work is the first to systematically compare the equivalence of WAA‑AS and WAA‑TENS in inflammatory muscle pain and to propose the circadian rhythm pathway as a common target. The findings provide valuable experimental evidence for non‑invasive, standardized physical analgesia with considerable translational potential. We commend the authors for their rigorous experimental design and meaningful findings. Nevertheless, several methodological and mechanistic considerations merit further discussion.

First, the sample size is relatively small and lacks an independent validation cohort. The study used only four rats per group (n=3 for some experiments). Although the authors calculated the required sample size based on assumed parameters, the relatively large standard error for certain measures suggests that inter‑individual variability may compromise statistical robustness. More importantly, transcriptomic sequencing and subsequent RT‑qPCR validation were performed on the same animal samples, rather than on an independent validation cohort, which may lead to overestimation of false‑positive rates among differentially expressed genes.

Second, the assessment of autophagic flux and collagen dynamics is incomplete. The study found elevated TGF‑β but reduced collagen deposition after WAA interventions, which the authors explain by the anti‑inflammatory role of TGF‑β in the acute phase. However, TGF‑β is also a well‑known driver of fibrosis, and its biphasic functions—anti‑inflammatory in the acute phase versus pro‑fibrotic in the chronic phase—are highly context‑dependent and may vary across species, tissue types, and injury models.² In the present study, only a 3‑day time point was examined; later time points (e g, day 7, day 14) were not evaluated Therefore, it remains unknown whether prolonged WAA‑TENS application might lead to delayed excessive collagen deposition Moreover, changes in autophagic flux during skeletal muscle repair are crucial for clearing damaged organelles and regulating inflammatory responses, yet this study did not measure autophagy‑related proteins (e g, LC3‑II/I, p62) or autophagic flux.

Third, a causal relationship between circadian genes and analgesic effects has not been established. Transcriptomic analysis revealed differential expression of Cry1, Per2, Nfil3, and Nr1d2, with distinct regulation patterns between the two interventions. However, these data are only correlative and do not provide causal evidence. For example, knockout or pharmacological inhibition of Per2 or Cry1 using CRISPR‑Cas9 followed by WAA intervention—if the analgesic effect is abolished or attenuated—would directly demonstrate that circadian genes mediate WAA’s actions. Additionally, the study did not measure key circadian output molecules (e g, melatonin, corticosterone) or behavioral rhythms (e g, activity‑rest cycles), limiting interpretation of the relationship between peripheral local clocks and the central clock.

Fourth, the stimulation parameters of WAA‑TENS and their clinical equivalence require further optimization. The authors used a dense‑sparse wave (2 Hz/100 Hz, 1 mA current, 20 minutes per session, once daily for 3 days) and demonstrated equivalence to WAA‑AS. However, the rationale for selecting these specific parameters is not fully explained. Parameters such as frequency, intensity, treatment duration, and frequency of sessions may all influence analgesic effects and circadian gene expression patterns. Furthermore, although WAA‑TENS has the advantage of being non‑invasive and standardized, the interventions were performed under anesthesia (tribromoethanol was used for both modeling and pre‑intervention procedures), which may affect neural conduction of electrical stimulation and the animal’s stress state.

Fifth, the crosstalk between inflammation and the circadian pathway deserves deeper analysis. The KEGG enrichment analysis identified both “circadian rhythm” and immune‑related pathways such as “antigen processing and presentation” and “Th1/Th2/Th17 differentiation”, but the interaction between these pathways was not further analyzed. It is well known that circadian genes directly regulate immune cell chemotaxis, cytokine secretion, and antigen presentation.³ Emerging evidence suggests that the TGF‑β signaling pathway itself may be controlled via a clock‑dependent mechanism, particularly in the context of inflammatory and fibrotic diseases.⁴ Notably, TGF‑β1 has been shown to promote inflammatory resolution by regulating macrophage polarization in acute injury models.⁵ Furthermore, the crosstalk between circadian rhythm and Th17 cell differentiation is increasingly recognized, with NFIL3 and RORA acting as key molecular bridges.⁶ A co‑expression network analysis or pathway crosstalk analysis to reveal the strength of association between circadian genes and immune pathway genes would provide deeper insight into the integrated regulatory mechanisms of WAA.

In conclusion, Mai et al have provided the first evidence of equivalence between WAA‑TENS and traditional WAA‑AS in an acute inflammatory muscle pain model, and have innovatively linked the circadian pathway to the analgesic mechanism of wrist‑ankle acupuncture. Our suggestions are not meant to negate the current conclusions, but rather to offer constructive directions for future research in this field. We sincerely look forward to further mechanistic validation by the authors, which will facilitate the translation of wrist‑ankle acupuncture and its derivative techniques into standardized, non‑invasive clinical therapies.

Disclosure

The authors report no conflicts of interest in this communication.