Applying RNA Sequencing Technology to Explore Repair Mechanism of Tuina on Gastrocnemius Muscle in Sciatic Nerve Injury Rats
Peripheral nerve injury (PNI) refers to damage to the peripheral nerve plexus, nerve trunk, or its branches caused by external forces. Skeletal muscle-derived cells play a significant role in nerve repair due to their strong secretory function and ability to differentiate into Schwann cell-like cells or other cell types, thereby promoting peripheral nerve regeneration. Tuina, a traditional Chinese manual therapy, has been shown to alleviate symptoms such as muscle spasms and pain caused by nerve damage. This study aimed to explore the effects of Tuina on the recovery of sciatic nerve injury (SNI) in rats, focusing on changes in hind limb muscle strength and genetic alterations in the gastrocnemius muscle using RNA sequencing technology.
Experimental Design and Methodology
The study involved 27 male Sprague-Dawley (SD) rats aged 6 weeks, weighing 200 ± 10 g. The rats were randomly divided into three groups: the Sham group, SNI group, and Tuina group. Before surgery, the rats were fasted and deprived of water for 24 hours and then anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (0.35 mL/100 g). The rats were fixed in a prone position, and the right hip-femoral junction was prepared with iodophor, shaving, and re-sterilization. A 1 cm incision was made along the direction of the sciatic nerve, exposing the lower edge of the piriformis. The sciatic nerve was identified, sterilized, and sutured in the Sham group. In the SNI and Tuina groups, the sciatic nerve was clamped with hemostatic pliers 5 mm distal to the nodule for 5 seconds with full force (6 N), creating a 2 mm injury point. The area was then flushed with saline, and the wound was sterilized and sutured.
Post-surgery, the hind limbs of the Sham group rats were able to move freely within a day. In contrast, the SNI and Tuina group rats exhibited straightened knee joints with limited flexion and limping, indicating successful modeling of sciatic nerve injury. The rats in the Sham and SNI groups were fed routinely, while the Tuina group began treatment on the 7th day post-surgery. Tuina techniques, including point pressing, plucking, and kneading, were applied to the Yinmen (BL37), Chengshan (BL57), and Yanglingquan (GB34) points on the affected side once daily for 9 minutes. The treatment was administered for 10 consecutive days, followed by a 1-day rest, and repeated for another 10 days, totaling 20 treatments. To reduce stress, the rats were petted and stroked for 9 minutes before each intervention.
Behavioral Testing and Muscle Strength Assessment
An electrically inclined plate tester was used to measure changes in hind limb muscle strength. Behavioral testing was conducted at baseline before surgery and on days 0, 5, 10, 15, and 20 of the intervention. The rats’ heads were placed on the board, and the angle was gradually increased until the rats could no longer maintain their position for 5 seconds. The critical angle was recorded, and the average of three measurements was taken.
RNA Sequencing and Data Analysis
After the intervention, nine rats from each group were anesthetized, and the right gastrocnemius muscle tissue was collected. Total RNA was extracted from the tissue of three rats, mixed as a sample, and each group had three biological replicates. RNA sequencing was performed, and the data were analyzed using SPSS 22.0. One-way analysis of variance (ANOVA) was used to compare the groups.
Results
The inclined plate test results showed no significant differences in the angle at baseline among the groups. However, the angle decreased significantly in the SNI and Tuina groups compared to the Sham group before intervention (7 days post-surgery). By the 10th intervention, the Tuina group showed a significant increase in the inclined plate angle compared to the SNI group (P < 0.05), with further increases observed in the 15th and 20th interventions (P < 0.01). However, the Tuina group still showed significant differences compared to the Sham group.
RNA sequencing revealed 44 differentially expressed genes (DEGs) between the Sham and Tuina groups. Four genes in the Sham group and 20 genes in the Tuina group showed significant differences compared to the SNI group. Gene ontology (GO) analysis indicated that DEGs were involved in cellular processes, single-organism processes, metabolic processes, regulation of biological processes, response to stimuli, cell parts, cells, organelles, and binding. GO functions were enriched in the regulation of complement activation, response to stimuli, cellular senescence, and Wnt signaling pathway involved in digestive tract morphogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed involvement in amino acid metabolism, lipid metabolism, metabolism of cofactors and vitamins, xenobiotics biodegradation and metabolism, immune system, nervous system, immune diseases, and neurodegenerative diseases. KEGG pathways were enriched in cytokine-cytokine receptor interaction, chemical carcinogenesis, and AMPK signaling pathway.
Discussion
The inclined plate test results demonstrated that Tuina promoted recovery and regeneration of SNI, facilitated connections between injured nerves and target organs, and delayed gastrocnemius muscle atrophy. The DEGs analysis highlighted that Ankrd1 (ankyrin repeat domain 1) had the largest up-regulated expression in GO enrichment, while Eda2r (ectodysplasin A2 receptor) showed the largest up-regulated expression in KEGG enrichment. IL12rb2 (interleukin 12 receptor subunit beta 2) exhibited the largest differential expression in GO function and KEGG pathway enriched down-regulated expression.
Ankrd1 is a protein involved in regulating muscle fiber type conversion. Its constituent cell components include Band I, and Tuina intervention may adjust the structure of myofilaments, regulate skeletal muscle differentiation, and respond to muscle strain, thereby stabilizing the gastrocnemius morphology and promoting nerve repair through Ankrd1 mRNA expression in the gastrocnemius muscle’s Band I. IL12rb2 plays a crucial role in PNI repair, while Eda2r promotes nerve repair by regulating the NF-kB, JNK, and p53 signaling pathways related to analgesia.
Conclusion
Tuina promoted the recovery of hind limb muscle strength in SNI rats, potentially by improving gastrocnemius gene expression. The DEGs involved multiple biological functions and pathways, suggesting that Tuina’s repair mechanism may be related to cellular processes, single-organism processes, response to stimuli, and cytokine-cytokine receptor interaction, AMPK, and other signaling pathways. The regulation of gene expression of Ankrd1, IL12rb2, and Eda2r in the gastrocnemius muscle may also contribute to the repair of SNI in rats.
doi.org/10.1097/CM9.0000000000001960
Was this helpful?
0 / 0