Study On The Mechanism Of Regulating Pain Sensitization In Rats With Sciatic Nerve Injury By Needling Huantiao Acupoin

Purpose:Through research methods such as behavior,morphology,molecular biology,proteomics,etc.From the perspective of inhibiting the expression of inflammatory factors in the injured site,dorsal root ganglia,spinal dorsal horn,and the activation of satellite glia and astrocyte,revealing the mechanism of peripheral and central pain sensitization caused by sciatic nerve injury,and the mechanism by which triple puncture the "Huantiao" acupoint reduces the degree of peripheral and central pain sensitization,Provide new theoretical support for clinical acupuncture treatment of pain.Material and method:1.Animal grouping and modelingSeventy SPF grade SD rats were divided into sham operation group(SHAM),model group(SNCI),triple acupuncture group(TA),single acupuncture group(SA)and drug group(Xi Yao Group,YW),with 14 rats in each group.In addition to SHAM group,the other 4groups were used to prepare the rat sciatic nerve crush injury model by classical clamp method.After successful modeling,2 rats in each group were randomly selected for nerve conduction velocity detection.Except for the sham operation group,the nerve conduction velocity in other groups was below 10 m/s,which means that the modeling of this group was successful.Before treatment on the second day of modeling,the thermal shrinkage threshold was detected and recorded.2.Intervention methodsAfter 1 day of modeling,each group began acupuncture treatment according to the corresponding intervention measures.In TA and SA groups,the "Huantiao" point on the same side of the model was selected as the treatment site.In SA group,the depth of acupuncture was about 10～12 mm,and the depth was appropriate to touch the deep muscle tissue below the injection site;In TA group,first prick a needle at the middle of the acupoint,with a depth of 10～12 mm,and then prick a needle at 5mm on both sides of the needle along the line of the gallbladder meridian,with the needle tip pointing to the focus.The two groups were treated once a day for 20 minutes each time for 14 consecutive days.The drug group was treated with ibuprofen suspension 0.5 mg/kg body weight by gavage once a day for 14 consecutive days.3.Sampling and index detectionAll experimental rats were evaluated for the thermal contraction threshold and the sciatic nerve motor function before the material was taken,and then the electrophysiological test was performed under anesthesia,and blood was taken from the abdominal aorta.After the rats were killed under excessive anesthesia,the sciatic nerve was taken.HE staining was used to observe the histopathological changes of sciatic nerve in rats with sciatic nerve injury;Detection of IL-1β、IL-6、TNF-α、PGE2 content in serum by ELISA;Detection of IL-1β、IL-6、TNF-α、PGE2 content in sciatic nerve tissue by ELISA.Six rats were randomly selected from each group,and the dorsal root ganglion of rats was taken.The content of IL-1β、IL-6and TNF-α content in dorsal root ganglion of rats with sciatic nerve injury was detected by ELISA.ATP content in dorsal root ganglion tissue was measured by colorimetry.The expression of Cx43、GFAP、P2X7 in dorsal root ganglion tissue was detected by RT-PCR.The remaining 6 rats in each group were selected and the spinal cord tissues of rats were taken.The content of IL-1β、IL-6 and TNF-α in the spinal cord of rats with sciatic nerve injury was detected by ELISA;ATP content in the spinal cord of rats was measured by colorimetry.The expression of Cx43、GFAP、P2X7 in spinal cord tissue was detected by RT-PCR;The expression levels of Cx43、GFAP、P2X7 in the spinal cord of rats with sciatic nerve injury were detected by immunohistochemistry and immunofluorescence.Results:Part 1:Through the measurement of pain behavior,histomorphology and inflammatory factors,the effectiveness of triple needling of "Huantiao" acupoint in alleviating the pain degree of rats with sciatic nerve injury was clarified.1.General state observation of rats: no rats died during the experiment.The rats in SHAM were in good condition,with normal water intake and bowel movement.The rats in SNCI were lame and easily excited.Compared with SNCI,the above performance of the rats in TA、SA and YW was improved to varying degrees.2.Evaluation of motor function of sciatic nerve in experimental ratsThe sciatic nerve function index(SFI)of rats with crush injury prepared by clamp method in SNCI decreased significantly(P<0.01).After treatment,compared with SNCI,the level of increase in TA,SA and YW increased significantly(P<0.01),and the level of increase in TA was higher than that in SA and YW(P<0.01).3.The evaluation results of the latent period of the foot contraction reflex caused by radiation heat stimulation show:The results showed that before treatment,compared with the SHAM group,the PWL of rats with crush injury prepared by clamp method was significantly decreased(P<0.01),and there was no significant difference between the different modeling groups(P>0.05),which was comparable between groups,suggesting that this modeling method caused serious damage to the neuromotor function of rats.After treatment,compared with SNCI group,the PWL of TA group increased significantly(P<0.01),and the effect was better than that of SA group and YW group.It suggested that the effect of triple needling was obvious in improving the motor function of sciatic nerve.4.HE staining results of sciatic nerve tissue in experimental ratsThe sciatic nerve tissue of rats in SHAM group was normal,the cells were flat and orderly arranged,without hyperemia and edema.In SNCI group,hyperemia and edema were observed in the sciatic nerve tissue of rats,with disordered cell arrangement.A small amount of inflammatory cells were seen in TA group,SA group and YW group,and the cell arrangement was slightly disordered.TA group was superior to SA group and YW group.5.The content of IL-1β、IL-6、TNF-α、PGE2 in serum of rats in each group was detected by ELISA.The results showed that:Compared with SHAM group,the level of IL-1β、IL-6、TNF-α、PGE2 in SNCI group was significantly higher(P<0.01).Compared with SNCI group,the level of IL-1β、IL-6、TNF-α、PGE2 in TA group,SA group and YW group decreased significantly(P<0.01),and the level of IL-1β、IL-6、TNF-α、PGE2 in TA group was lower than that in SA group and YW group(P<0.01).6.The content of IL-1β、IL-6、TNF-α、PGE2 in the sciatic nerve tissue of rats in each group was detected by ELISA.The results showed that:Compared with SHAM group,the level of IL-1β、IL-6、TNF-α、PGE2 in SNCI group was significantly higher(P<0.01).Compared with SNCI group,the level of IL-1β、IL-6、TNF-α、PGE2 in TA group,SA group and YW group decreased significantly(P<0.01),and the level of IL-1β、IL-6、TNF-α、PGE2 in TA group was lower than that in SA group and YW group(P<0.01).Part 2:Based on the activation of satellite glia,the discusse of mechanism of regulating peripheral sensitization of rats with sciatic nerve injury by triple needling of "Huantiao" acupoint.1.The content of IL-1β、IL-6 and TNF-α in the dorsal root ganglion of rats in each group was detected by ELISA.The results showed that:Compared with SHAM group,the level of IL-1β、IL-6 and TNF-α in SNCI group was significantly higher(P<0.01).Compared with SNCI group,the level of IL-1β、IL-6 and TNF-α in TA group,SA group and YW group decreased significantly(P<0.01),and the level of IL-1β、IL-6 and TNF-α in TA group was lower than that in SA group and YW group(P<0.01).2.Determination of ATP content in rat dorsal root ganglion tissue by colorimetry.Compared with SHAM,the ATP content in the dorsal root ganglion tissue of SNCI,TA,SA,and YW groups significantly increased(P<0.01);Compared with SNCI,the ATP content in the dorsal root ganglion tissue of rats in TA,SA,and YW groups decreased significantly(P<0.01);The ATP content in the dorsal root ganglion tissue of rats in TA group was lower than that in SA group and YW group(P<0.01).3.RT-PCR method was used to detect the expression of Cx43、GFAP、P2X7 in dorsal root ganglion of rats in each group.The results showed that:Compared with SHAM group,the levels of Cx43、GFAP、P2X7 in SNCI group were significantly higher(P<0.01).Compared with SNCI group,the levels of Cx43、GFAP、P2X7in TA group,SA group and YW group were significantly lower(P<0.01),and the levels of Cx43、GFAP、P2X7 in TA group were lower than those in SA group and YW group(P<0.01).Part 3:Based on the activation of astrocytes,the discusse of mechanism of triple needling of "Huantiao" acupoint regulating central sensitization in rats with sciatic nerve injury.1.The content of IL-1β、IL-6 and TNF-α in spinal dorsal horn tissue of rats in each group was detected by ELISA.The results showed that:Compared with SHAM group,the level of IL-1β、IL-6 and TNF-α in SNCI group was significantly higher(P<0.01).Compared with SNCI group,the level of IL-1β、IL-6 and TNF-α in TA group,SA group and YW group decreased significantly(P<0.01),and the level of IL-1β、IL-6 and TNF-α in TA group was lower than that in SA group and YW group(P<0.01).2.Determination of ATP content in rat spinal dorsal horn by colorimetryCompared with SHAM,the ATP content in the spinal dorsal horn of rats in SNCI,TA,SA,and YW groups significantly increased(P<0.01);Compared with SNCI,the ATP content in the spinal dorsal horn of rats in TA group,SA group,and YW group decreased significantly(P<0.01);The ATP content in the spinal dorsal horn of rats in TA group was lower than that in SA group and YW group(P<0.01).3.RT-PCR method was used to detect the expression of Cx43、GFAP、P2X7 in the spinal dorsal horn of rats in each group.The results showed that:Compared with SHAM group,the levels of Cx43、GFAP、P2X7 in SNCI group were significantly higher(P<0.01).Compared with SNCI group,the levels of Cx43、GFAP、P2X7in TA group,SA group and YW group were significantly lower(P<0.01),and the levels of Cx43、GFAP、P2X7 in TA group were lower than those in SA group and YW group(P<0.01).4.The expression level of Cx43、GFAP、P2X7 protein in the spinal dorsal horn of rats in each group was detected by immunohistochemistry.The results showed that:Compared with SHAM group,the level of Cx43、GFAP、P2X7 in SNCI group was significantly higher(P<0.01).Compared with SNCI group,the level of Cx43、GFAP、P2X7 in TA group,SA group and YW group was significantly lower(P<0.01),and the level of Cx43、GFAP、P2X7 in TA group was lower than that in SA group and YW group(P<0.01).5.Immunofluorescence assay was used to detect P2X7、Cx43 and GFAP protein expression markers in spinal dorsal horn tissue of rats in each group:The fluorescence intensity of P2X7,Cx43,and GFAP proteins in the dorsal horn of the rat spinal cord was higher in the SNCI group than in the SHAM group.Compared with the SNCI group,the fluorescence in TA group,SA group,and YW group decreased or decreased to varying degrees.TA group was superior to SA group and YW group.Conclusion:1.Triple needling of "Huantiao" acupoint can improve the general state of rats with sciatic nerve injury,reduce the pain threshold of foot contraction caused by radiation heat stimulation in rats with sciatic nerve injury,improve the pathological morphology of sciatic nerve tissue in rats with sciatic nerve injury,inhibit the expression of IL-1β、IL-6、TNF-α、PGE2 inflammatory factor in serum and nerve tissue of rats with sciatic nerve injury,reduce inflammatory reaction,and improve the pain symptoms of rats with sciatic nerve injury.It is effective to treat sciatica in rats with sciatic nerve injury by triple needling of "Huantiao".2.Triple needling of "Huantiao" acupoint may inhibit the release of ATP from small and medium-sized neurons in the dorsal root ganglion of rats,reduce the expression of P2X7 receptor and Cx43 half channel in satellite glia,inhibit the activation of satellite glia and the expression of pro-inflammatory factors,and reduce the peripheral sensitization of sciatica.3.Triple needling of "Huantiao" acupoint may inhibit the release of ATP from synaptic sites in the spinal cord of rats,reducing P2X7 mediated release of inflammatory factors from microglia,thereby reducing the overexpression of Cx43 half channels,inhibiting astrocyte activation and inflammatory factor expression,and reducing the degree of axis sensitization in sciatica.