| Objective:(1)To analyze the protein of Shaoyao Gancao decoction with significant changes in dorsal root ganglion in chronic inflammatory pain.(2)To explore the mechanism of dorsal root ganglion Sema3 G,IL-6 and CCL2 in the treatment of chronic inflammatory pain by Shaoyao Gancao decoction.Methods:Experiment 1 18 SD rats were randomly divided into control group,model group and Shaoyao Gancao decoction group,with 6 rats in each group.A model of Chronic Inflammatory pain(CIP)induced by subcutaneous injection of plantar of100μl Complete Freund’s(CFA)was used in model group and Shaoyao Gancao decoction group and the control rats with the 100μl normal saline.Rats in Shaoyao Gancao decoction group was fed with Shaoyao Gancao decoction 24 hours after operation,once a day,for 7 consecutive days.The rats in model group was given normal saline intragastric administration 24 hours after operation,once a day,for 7consecutive days.The control group was equally grasped without any intervention.Paw withdrawal threshold was measured by Von-frey painometer before intervention and on the 1st,3rd,5th and 7th day after intervention.Paw withdrawal latency pain threshold was measured with thermosensitive pain meter before surgery and 3rd,5th and 7th days after surgery.Comprehensive protein expression of rat dorsal root ganglion was detected by total protein 4D Label free quantitative proteomics on the 7th day after surgery.Western blotting was used for the determination of expression of Sema3 G at protein level in rat dorsal root ganglion.The relative expression levels of IL-6 and CCL2 mRNA in rat dorsal root ganglion were detected by real-time PCR.Experiment 2 18 SD rats were randomly divided into control group,model+r AAV-h Syn-EGFP group,model + r AAV-h Syn-r Sema3g-P2A-EGFP group.Rats in control group were fed normally 4 weeks before modeling without intrathecal injection.Rats in model + r AAV-h Syn-EGFP group were intrathecally injected with60 ul r AAV-h Syn-EGFP 4 weeks before modeling.Rats in model + r AAV-h Synr Sema3g-P2A-EGFP group were intrathecally injected with 60μl Sema3 G agonist 4weeks before modeling.Modeling was performed after 4 weeks of intrathecal injection.Chronic inflammatory pain model induced by subcutaneous injection of plantar of 100μl CFA was used in model + r AAV-h Syn-EGFP group and model + r AAV-h Syn-r Sema3g-P2A-EGFP group and the control rats with the 100μl normal saline.Paw withdrawal threshold was measured before the operation and on the 1st,3rd,5th and 7th day after operation.Paw withdrawal latency was measured before the operation and on the 1st,3rd,5th,7th days after operation.The relative expression levels of IL-6 and CCL2 mRNA in rat dorsal root ganglion were detected by quantitative real-time PCR on the 7th day after operation.Result:Experiment 1: Effects of Shaoyao Gancao decoction on behavior result,the expression of comprehensive protein and Sema3 G of dorsal root ganglion,relative expression levels of IL-6 and CCL2 mRNA in rats with chronic inflammatory pain.(1)Effect of Shaoyao Gancao decoction on Paw withdrawal threshold in rats with chronic inflammatory pain: On the 7th day after surgery,the paw withdrawal threshold of model group was significantly lower than that of control group(P<0.05).Compared with model group,paw withdrawal threshold of Shaoyao Gancao decoction group was significantly increased(P<0.05).(2)Effect of Shaoyao Gancao decoction on Paw withdrawal latency in rats with chronic inflammatory pain: On the 3rd,5th day after surgery,the paw withdrawal latency of model group was significantly lower than that of control group(P<0.05);Compared with model group,paw withdrawal latency of Shaoyao Gancao decoction group was significantly increased(P<0.05).(3)Effect of Shaoyao Gancao decoction on quantitative proteomic analysis in rats with chronic inflammatory pain: On the 7th day after surgery,compared with control group,the 52 kinds of proteins were significantly down-regulated,and 17 kinds of proteins were significantly up-regulated in the dorsal root ganglion of the rats in the model group(P<0.001);Compared with model group,the 515 kinds of proteins were significantly down-regulated,and 21 kinds of proteins were significantly up-regulated in the dorsal root ganglion of the rats in the Shaoyao Gancao decoction group(P<0.001).(4)Effect of Shaoyao Gancao decoction on relative expression level of Sema3 G protein in DRG of rats with chronic inflammatory pain: On the 7th day after surgery,compared with control group,the expression of Sema3 G in dorsal root ganglion at protein level was significantly down-regulated in the model group(P<0.05).The expression of Sema3 G in dorsal root ganglion at protein level was significantly up-regulated in the Shaoyao Gancao decoction group(P<0.05).(5)Effect of Shaoyao Gancao decoction on relative expression level of IL-6 and CCL2 mRNA in DRG of rats with chronic inflammatory pain: Compared with control group,the relative expression level of IL-6 mRNA in model group was significantly increased(P<0.05);Compared with model group,the relative expression level of IL-6mRNA in Shaoyao Gancao decoction group was significantly decreased(P<0.05),and the relative expression level of CCL2 mRNA in model group was significantly increased(P<0.05).Compared with model group,the relative expression level of CCL2 mRNA in Shaoyao Gancao decoction group was significantly decreased(P<0.05)Experiment 2: Effects of intrathecal injection of r AAV-h Syn-r Sema3g-P2A-EGFP on behavior result,the relative expression levels of IL-6 and CCL2 mRNA in dorsal root ganglion of chronic inflammatory pain rats.(1)Effect of r AAV-h Syn-r Sema3g-P2A-EGFP on Paw withdrawal threshold in rats with chronic inflammatory pain:After modeling,the PWT in model + r AAV-h Syn-EGFP group was significantly lower than that in control group(P<0.05);On days 7after modeling,the PWT in model + r AAV-h Syn-r Sema3g-P2A-EGFP group was significantly higher than that in model + r AAV-h Syn-EGFP group(P<0.05).(2)Effect of r AAV-h Syn-r Sema3g-P2A-EGFP on Paw withdrawal latency in rats with chronic inflammatory pain:After modeling,the PWL in model +r AAV-h Syn-EGFP group was significantly lower than the control group(P<0.05);On days 3rd,5th and 7th after modeling,the PWL in model + r AAV-h Synr Sema3g-P2A-EGFP group was significantly higher than the model +r AAV-h Syn-EGFP group(P<0.05).(3)Effect of r AAV-h Syn-r Sema3g-P2A-EGFP on relative expression level of IL-6 and CCL2 mRNA in DRG of rats with chronic inflammatory pain:Compared with control group,the relative expression level of IL-6 mRNA in model +r AAV-h Syn-EGFP group was significantly increased(P<0.05).Compared with model+ r AAV-h Syn-EGFP group,the relative expression level of IL-6 mRNA in model +r AAV-h Syn-r Sema3g-P2A-EGFP group was significantly decreased(P<0.05).Compared with control group,the relative expression level of CCL2 mRNA in model+r AAV-h Syn-EGFP group was significantly increased(P<0.05).Compared with model + no-virus group,the relative expression level of CCL2 mRNA in model +r AAV-h Syn-r Sema3g-P2A-EGFP group was significantly decreased(P<0.05).Conclusion:(1)Shaoyao Gancao decoction has a specific therapeutic effect on chronic inflammatory pain rats,which can reduce the Paw withdrawal threshold and Paw withdrawal latency.(2)Shaoyao Gancao decoction may improve Paw withdrawal threshold and Paw withdrawal latency in rats with chronic inflammatory pain by upregulating the expression of Sema3 G and inhibit the relative expression levels of IL-6 mRNA and CCL2 mRNA in the dorsal root ganglia. |
PDF Full Text Request