Electroacupuncture Attenuates Inflammatory Pain Via Inhibition Of Chemokine CX3CL1 In Spinal Cord

Chronic pain is a series of disorder which is characterized by a long lasting intractable pain. It not only tortures the patients' physical and mental health, but also impairs their quality of life. In the United States, the economic loss caused by chronic pain estimates at 60 million dollars annually, which brings heavy burden to families and society. At present, analgesic drugs applied in clinic are mainly non-steroidal anti-inflammatory drugs(NSAIDs) and opioids, which have limited analgesic effect but strong side-effects like drug addiction. Considering the poor effects of productions in basic and clinical translational research, it is imperative to find a highly efficient way without side effects for chronic pain relief. Due to its safety, high efficacy at low cost, acupuncture is recognized as a viable alternative therapy in chronic pain treatment. Electroacupuncture(EA) is a novel therapeutic method based on traditional acupuncture and combined with modern electrotherapy. Concomitant with increasing use of the EA, although its analgesic effect is well documented, little is understood about its biological basis.Researches have been performed on its mechanisms and data from these studies have accumulated. EA exerts its analgesic effect via regulating the synthesis and release of kinds of bioactive substances. In addition, more evidences reveal that glial cells also play an important role in formation and maintenance of pain. If EA inhibits pain by regulating the information transferred between neurons and glia cells is worth exploring. Through literature review, we lock on the chemokine CX3CL1 and its receptor CX3CR1 as the target. After Cathepsin S induces increasing of CX3CL1 in spinal cord dorsal horn, its receptor CX3CR1 expressed in microglia could be subsequently activated, leading to sequential activation of p38 MAPK pathway and release of proinflammatory cytokines, such as IL-1?, IL-6 and TNF-? that induce the formation of pain.Since it remains unclear that whether EA restrains pain through interfering with the crosstalk between neuron and glia, we design the experiments to investigate whether EA suppresses pain via regulating CX3CL1/CX3CR1 interaction. The results may provide novel experimental evidences to illustrate the mechanisms of acupuncture analgesia and propel its clinical application. Experiment 1: Analgesic effect of electroacupuncture on inflammatory pain induced by complete Freund's adjuvantObjective: To observe the analgesic effect of electroacupuncture on inflammatory pain induced by complete Freund's adjuvant(CFA) intraplantar injection in rats.Methods: 40 adult male Sprague-Dawley(SD) rats(weight 180-200g) were randomly divided into 4 groups: Control group, CFA group, EA group and Sham group(n=10 per group). The pathological inflammatory pain models were induced by unilateral subcutaneous injection of CFA into the plantar surface of hindpaws of the animals in CFA, EA and Sham group. Animals in EA group received EA treatment at bilateral Zusanli acupoint respectively at 1, 3, 5, 7 days after models were set up. Animals in Sham group received EA treatment at bilateral inguinal area where no acupoint exists at the same time. Paw withdrawal mechanical threshold(PWMT) and paw withdrawal thermal latency(PWTL) were measured one time a day after CFA injection.Results: After CFA intraplantar injection, the pain thresholds of animals in CFA, EA and Sham group decreased significantly compared with Control group, with the appearance of mechanical and thermal hyperplasia(P < 0.01), and this effect lasted more than 7 days. With the EA treatment, hyperplasia was partly inhibited in animals of EA group, the pain thresholds increased visibly compared with CFA group(P < 0.05). Meanwhile, the pain thresholds of Sham group showed no statistical difference with the thresholds of the CFA group. Experiment 2: Effect of electroacupuncture on the expression and activation of CX3CL1/CX3CR1 and their downstream moleculesObjective: To illustrate the effect of CFA injection and EA treatment on expression and activation of certain molecules, including CX3CL1/CX3CR1, p38 MAPK and proinflammatory cytokines.Methods: The random grouping method was the same as the previous experiment and 60 adult male SD rats(weight 180-200g) were randomly divided into 4 groups(n=18 per group). Based on the results of Experiment 1, animals in each group were executed 1 and 3 days after the models were made. Tissue of lumbar enlargement of the spinal cords was collected after saline perfusion through the hearts. Western blot was used to detect the alternation of CX3CL1, CX3CR1 and p38 MAPK. The levels of IL-1?, IL-6 and TNF-? were tested with ELISA kits. At the same time, immunofluorescence(IF) staining was implemented to provide more evidences.Results:1. The expression of CX3CL1 in spinal cord was increased significantly because of CFA injection(P < 0.05), and EA treatment could inhibit this effect. Meanwhile, the expression of CX3CR1 was not affected neither by CFA injection nor EA treatment.2. As downstream molecule of CX3CL1/CX3CR1, p38 MAPK's expression did not change due to experimental intervention. But at 3 days after models were made, the expression of phosph-p38 MAPK was promoted(P < 0.05), indicated that p38 MAPK was abnormally activated. EA treatment, however, down-regulated the expression of phosphp38 MAPK, made it at the same level as the Control group.3. The levels of IL-1?,IL-6 and TNF-? were elevated due to p38 MAPK mediation in CFA group(P < 0.05). But the effects were inhibited in EA group. Experiment 3: CX3CL1 is a key factor in anti-inflammatory and analgesic effect of electroacupuncture.Objective: To verify EA exert anti-inflammatory and analgesic effects through modulating the expression of CX3CL1.Methods: Lumbosacral intrathecal catheters were constructed and implanted to male SD rats(weight 140-160g). 36 rats were selected and randomly separated into Ig G group, Ig G+EA group and FKN+EA group(n=12 per group).All experimental animals received intraplantar injection of CFA to set up pathological pain model. Animals in Ig G+EA group and FKN+EA group received EA treatment at bilateral Zusanli acupoint at 1 and 3 days after models were made. Control Ig G(for Ig G and Ig G+EA group) and CX3CL1(for FKN+EA group) were injected intrathecally. Behavioral tests and Western blot were then conducted to detect the relation between analgesic effect of EA and CX3CL1 expression level.Results:1. Mechanical and thermal hyperplasia were found in experimental animals in each group. Pain thresholds of Ig G+EA group were much higher than that of Ig G group due to EA treatment(P < 0.01). Whereas the analgesic effect of EA in FKN+EA group was reversed by CX3CL1 injection(P < 0.05).2. The expression of p38 MAPK still remained the same among 3 groups, but the level of phosphorylation differed a lot. The expression of phosph-p38 MAPK in FKN+EA group was higher than Ig G+EA group but lower than Ig G group(P < 0.05).Conclusions: 1. EA treatment could alleviate the mechanical and thermal hyperplasia caused by CFA injection, and has an analgesic effect. 2. The expression of CX3CL1 in spinal cord was elevated by set up of CFA inflammatory pain model. Phosphorylation level of p38 MAPK as well as release of proinflammatory cytokines was also promoted. EA treatment could restrain such changes. 3. EA treatment could inhibit the inflammation reaction mediated by CFA through regulating the expression of CX3CL1.Summary: The experiments used inflammatory pain model to detect the analgesic effect of EA treatment through behavioral tests, found that EA treatment could effectively increase the pain thresholds of rats. The experiments in morphology and molecular biology methods revealed that the down-regulation of CX3CL1 and inhibition of inflammatory reaction downstream were associated with EA treatment, indicated the cause-effect relationship between the two incidents. At last, with the combination of intrathecal administration and electroacupuncture treatment, the results proved that EA mediate analgesic effect via inhibiting the expression of CX3CL1 at spinal cord. The current study enriches the theories of acupuncture analgesia and provides important support for understanding the mechanisms of acupuncture.