| Objective:To study and observe the effects of modified Shengjiangsan on the pain behavior and memory function of rats with chronic constriction injury of the sciatic nerve(iCCI)and to study the effect of IL-6 and TNF-α in the spinal cord of rats with the lifting and dispersing of the rats from the p38MARK signaling pathway, while to probe into the mechanism and therapeutic target of modified Shengjiangsan in the treatment of neuropathic pain.Methods:1. Preparation of concentrate of modified Shengjiangsan:Rhubarb, Jiang Huang, Jiangcan, cicada, Rhizoma Corydalis, Radix Paeoniae Alba, Radix Glycyrrhizae preparata were taken while it were boiled and filtered. Then, the drug filtrate were collected and centrifuged. After centrifugal decompression, concentration of the concentrated liquid of modified Shengjiangsan and 4℃ for the preservation of the refrigerator.2. Effect of modified Shengjiangsan in the behavior of iCCI in rats with neuropathic pain and its memory function:65 SD male rats were randomly divided into 5 groups which the Sham operation group, model control group, low dose group, medium dose group and high dose treatment group, with 13 rats in each group. In the sham operation group, they were only exposed the right sciatic nerve but not ligation, and the other groups were established iCCI model. After seventh days of evaluation and after the success of the model, they were given the medicines while in the Sham operated group and model control group, they were treated with the same physiological saline. In the Low, medium and high dose treatment group, they were respectively given the 2.7g/kg,5.4g/kg, 10.8g/kg of modified Shengjiangsan of the stomach,2 times a day (8Am,4Pm)for 21 consecutive days. Before feeding and after feeding of 1d,3d,7d, 10d, L4D, 17D,21d, the spontaneous pain behavior score, and the threshold of pain threshold and heat pain threshold were respectively measured. Before feeding and after feedingof 7d, 10d, 1d,14d,17D,21d, the memory function were tested by the morris water maze test.3. Influence of modified Shengjiangsan in ICCI rat spinal dorsal horn inflammatory mediators:130 SD rats were randomly divided into 6 groups:the sham operation group, the model control group, the low dose group, the middle dose group and the high dose treatment group. And they were 5 rats in normal group, and 25 rats in each group. The normal group, they were not treated with surgery and drug treatment; and in the sham operation group, they were exposed to the right sciatic nerve but not ligation; The iCCI model of other groups were established.After seventh days of evaluation after the success of the model can be given the medicines, in the normal group, they were fed the normal feeding while in the sham operated group and model control group, they were treated with the same physiological saline. In the Low, medium and high dose treatment group, they were respectively given the 2.7g/kg,5.4g/kg,10.8g/kg of modified Shengjiangsan of the stomach,2 times a day (8Am,4Pm)for 21 consecutive days. Rats in each group were given the materials before feeding and after feeding 1D,7d,14d,21d, the amount of each material was 1 in the normal group and the amount of material of the other groups, with 5 in each groups. Then, the rat spinal cord L4 -6 segments were taken and the concentration of TNF-α and IL-6 in spinal cord were detected by the ELISA method;The expression level of TNF- and mRNA were detected by fluorescence quantitative PCR assay;The expression and phosphorylation level of p38MAPK protein were detected by Western-blot method.Outcome:1. spontaneous pain behavior score:The medicine after 10d,14d,17d, the spontaneous pain behavior score in the high dose treatment group was significantly lower than that in the model control group, and the difference had statistically significant.The medicine after 21d, the spontaneous pain behavior score in the high dose treatment group was significantly lower than that in the model control group, and the difference had statistically significant. The difference of three treatment groups in different feeding time had statistically significant.2. Mechanical pain threshold test results:Different time of sham operation group and the difference of each group had statistical significance. The medicine after 10d,17d,21d, the mechanical pain threshold in the low, middle and high dose treatment group were significantly higher than that in model control group, and the difference had statistically significant (P<0.01). There were significant differences in different time (P<0.05). Three treatment groups after feeding, the mechanical pain threshold in the high dose treatment group was the most obvious.3. Determination results of heat pain threshold:The medicine after 17d, 21d, compared with the low dose group and model group, heat pain threshold in the high dose group and middle dose group were significantly higher than in low dose group and the model control group, and the difference had statistically significant;There were significant differences in different time between each groups (P<0.05). In addition, the behavior detection results showed that modified Shengjiangsan have the effect of analgesia and the best analgesic effect is the high dose treatment group of 10.8g/kg.4. Experimental results of water maze:There was no significant difference in the different time, different dosage, platform crossing average between the groups (F=0.895, P=0.563), and it explained that the modified Shengjiangsan had no effect on the memory function of rats.5. Results of TNF-α and mRNA TNF-α in spinal cord:The medicine after 1d, 7d, the content of TNF-α in the spinal cord in the three treatment groups was significantly decreased than that in the sham operation group and the model group, and the differences had the statistical significance (P<0.01). The comparison of the levels of TNF-α alpha in the spinal cord of three treatment groups was high dose group>medium dose group>low dose group, and differences had no statistical significance in the pairwise comparison. In addition, the First days after feeding, in the high dose treatment group, the content of TNF-a in the spinal cord of the treatment group was the lowest, and the decrease was the most obvious, and slowly declined after a time which it explained that the modified Shengjiangsan had a role of TNF-a in the spinal cord in the early stage.6. Results of IL-6 content and mRNA IL-6 expression in spinal cord:The IL-6 content in different time model group and each group was significantly different than that in the spinal cord (P<0.01). IL-6 content in spinal cord of different time model group and each group had statistical significance (P<0.01). In addition, the three treatment groups after feeding, the content of IL-6 in the spinal cord was decreased, the comparison of IL-6 content in the spinal cord of the three treatment groups was that high dose group>medium dose group>low dose group, and differences had no statistical significance in the pairwise comparison. And the role of the IL-6 in the spinal cord in the continuous reduction of the spinal cord was explained.7. Results of p38 total protein and p-p38 protein content:Compared with normal group, the level of p38 phosphorylation in sham operation group and model group was increased;The level of p38 phosphorylation in middle and low dose treatment groups was maintained at elevated levels;The p38 phosphorylation level in high dose treatment group was increased after operation while was sustained reduction after feeding. Last, the p38 phosphorylation levels was closed in normal group.The experimental results explained that the modified Shengjiangsan can inhibit the p38MAKR pathway to achieve the effect of analgesia.Conclusion:1. The spontaneous pain behavior scores of rats in the iCCI model rats were significantly higher. The mechanical threshold value, the thermal pain threshold was significantly decreased, and it was maintained for a long time, thus it explain that the time of pain duration of iCCI model are long and the stability is good.2. The modified Shengjiangsan which can improve the pain behavior of neuropathic pain in iCCI rats. The analgesic effect are positively related to the concentration of the modified solution. In addition, the best analgesic effect are 10.8g/kg, bid, and there are no adverse effects on memory function of rats.3. The p38MAPK signal transduction pathway can be inhibited by the modified Shengjiangsan,and can reduce the expression of TNF-α, IL-6mRNA and protein in spinal cord, thus it relieved neuropathic pain in rats with iCCI effect. |
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