| Pur Pose:Oxaliplatin is a first-line chemotherapy drug for gastrointestinal malignant tumors.Its incidence of neuropathic pain(CINP)is 76%-90%.The pathogenesis is closely related to pain sensitization and nerve cell apoptosis.This disease belongs to the category of "blood paralysis" in Chinese medicine,and the exclusive prescription is Huangqi Guizhi Wuwu Decoction.In this study,the oxaliplatin-induced chemotherapy-induced neuropathic pain(CINP)rat model was used as the research object,using cellular biomolecule detection methods to detect that TNF-α,IL-1β,IL-6 inflammatory factors upstream of the MAPK pathway Expression,the expression of MAPK family members ERK1/2,p38 and JNK protein expression,the expression of key proteins downstream of the pathway c-Fos,CREB,Nf-κB protein expression.To explore the effect of Huangqi Guizhi Wuwu Decoction on reducing pain sensitization and anti-dorsal root ganglion injury,and then to elaborate the prevention and repair of Huangqi Guizhi Wuwu Decoction induced by oxaliplatin chemotherapy drugs The mechanism of action of CINP rat nerve cell injury provides a scientific basis for the clinical application of Huangqi Guizhi Wuwu Decoction in preventing and treating CINP.Methods:1.Experiment 1 Randomized controlled method was used to divide SPF 200-220 g and 45 female SD rats into blank group(without treatment),CINP model group(oxaliplatin chemotherapy model),and Western medicine prevention and treatment group(oxaliplatin chemotherapy model + duloxetine gavage),Huangqi Guizhi Wuwu Decoction gavage prevention and treatment group(oxaliplatin chemotherapy model+Huangqi Guizhi Wuwu Decoction),Huangqigui Zhiwuwu Decoction external treatment prevention and treatment group(Oxaliplatin Chemotherapy Model+Huangqi Guizhi Wuwu Decoction washing),9 rats in each group,continuous treatment for 15 days,and treatment at the same time on the specified date(Day 0,3,6,9,12,15)Rat weight,mechanical pain threshold,cold pain threshold,and hot pain threshold were measured.On the 15 th day,after fasting the rats but drinking water for 24 hours,anesthetize with 10% chloral hydrate(at 3 m L/kg body weight)for intraperitoneal injection.Then fix the rat supine position on the rat stand,prepare the skin to disinfect the abdominal skin of the rat with iodophor,cut the skin and peritoneum,reveal the abdominal aorta,and slowly draw blood from the syringe,and place the arterial blood in a 4 ℃ incubator for 2h,centrifuge at 2000 rpm for 15 min,and take the upper layer The light yellow liquid(serum)was placed in labeled sterile EP tubes and stored in a refrigerator at-80 ℃ for later use.In order to prevent the metal from contaminating the tissue,use a sterilized ceramic knife to cut off the excess nerves upstream and downstream of the required ganglion adjacent to the L4-L5 lumbar spine of the rat,obtain the nerve at the intervertebral foramen of the L4-L5,and obtain the dorsal root of the rat L4-L5 spinal cord There are a total of 4 pairs of ganglia,which are sealed with 1.5m L cryopreservation tubes and placed in a refrigerator at-80 ℃ to be stored frozen.Serum levels of TNF-α,IL-1β,IL-6 inflammatory factors in serum were measured by ELISA,and neuropathological morphology of rat L4-L5 was observed by HE staining Morphological observation of dorsal root ganglion.2.Experiment 2 Randomized controlled method was used to divide SPF 200-220 g and 30 female SD rats into blank group(without treatment),CINP model group(oxaliplatin chemotherapy model),and Western medicine prevention and treatment group(oxaliplatin chemotherapy model + duloxetine gavage),Huangqi Guizhi Wuwu Decoction gavage prevention and treatment group(oxaliplatin chemotherapy model+Huangqi Guizhi Wuwu Decoction),Huangqigui Zhiwuwu Decoction external treatment prevention and treatment group(Oxaliplatin Chemotherapy Model+Huangqi Guizhi Wuwu Decoction washing),6 rats in each group,continuous treatment for 15 days.On the 15 th day,after fasting the rats but drinking water for 24 hours,anesthetize with 10% chloral hydrate(at 3 m L/kg body weight)for intraperitoneal injection.Then fix the rat supine position on the rat stand,prepare the skin to disinfect the abdominal skin of the rat with iodophor,cut the skin and peritoneum,reveal the abdominal aorta,and slowly draw blood from the syringe.In order to prevent the metal from contaminating the tissue,use a sterilized ceramic knife to cut off the excess nerves upstream and downstream of the required ganglion adjacent to the L4-L5 lumbar spine of the rat,obtain the nerve at the intervertebral foramen of the L4-L5,and obtain the dorsal root of the rat L4-L5 spinal cord There are a total of 4 pairs of ganglia,which are sealed with 1.5m L cryopreservation tubes and placed in a refrigerator at-80 ℃ to be stored frozen.Western-blot detected ERK1/2,P38,JNK,c-Fos,CREB,Nf-κB protein expression in rat L4-L5 spinal dorsal root ganglia,IHC immunity Histochemical detection of L4-L5 spinal dorsal root ganglia showed ERK1/2,p38 and JNK protein expression.3.The experimental data of each group of rats was analyzed by SPSS 22.0 software.The data of each group was expressed as the mean ± standard deviation(x± s).The mean of multiple groups of measurement data was compared with the homogeneity of variance.Analysis,P <0.05 means that there is a significant difference,which has statistical significance.Results:1.The weight of rats in each group showed that at baseline(the 0th day),there was no statistical difference in the weight of rats in each group(P > 0.05);with the application of the chemotherapeutic drug oxaliplatin,it started on the 6th day Oxaliplatin-induced chronic CINP model rats,the western medicine duloxetine group,and Huangqi Guizhi Wuwu Decoction gavage control group reduced body weight growth rate,which was statistically different from the blank group rats(P < 0.05),the weight gain of rats in Huangqi Guizhi Wuwu Decoction gavage control group continued to the 12 th day,and the rats in the western medicine duloxetine group and the chronic CINP model group continued until the 15 th day;the western medicine duloxetine group had no effect on preventing and controlling the slow weight gain caused by oxaliplatin chemotherapy,and intragastric administration of Huangqi Guizhi Wuwu Decoction had a certain effect on preventing and controlling the slow weight gain caused by oxaliplatin chemotherapy,but Huangqi Guizhi Wuwu Decoction The effect of bubble washing has obvious advantages.The weight gain of this group of rats is compared with the blank group of rats at various time points(Day 0,3,6,9,12,15),and there is no statistical difference(P > 0.05).2.The results of the mechanical pain threshold of rats in each group showed that at baseline(the 0th day),there was no statistical difference in the mechanical pain threshold of rats in each group.With the application of the chemotherapeutic drug oxaliplatin,it started on 9th day the chronic CINP model group,the mechanical pain threshold gradually decreased until the 15 th day.The mechanical pain threshold was statistically different from the blank group at the time node(Day 9,12,15)(P < 0.05).Western medicine The duloxetine group,Huangqi Guizhi Wuwu Decoction gavage and bubble washing group had a certain effect on the prevention and treatment of mechanical pain threshold drop.Western medicine duloxetine and Huangqi Guizhi Wuwu Decoction have a certain therapeutic effect(the 12 th and 15 th days)when the oral administration of oxaliplatin-induced mechanical pain decreases,but the preventive effect between the two is There was no significant difference at each time point;Huangqi Guizhi Wuwu Decoction external washing has a good effect on the prevention and treatment of oxaliplatin-induced decrease of pain threshold.The mechanical pain threshold of rats is at multiple time points(Day 3,6,12,15)No decline,compared with the blank group of rats,there is no statistical difference(P > 0.05),the prevention and treatment effect is better than the western medicine duloxetine and Huangqi Guizhi Wuwu Decoction,in(Day 12,15),there was statistical difference(P < 0.05).3.The pain threshold of rats in each group showed that at baseline(the 0th day),there was no statistical difference in the cold pain threshold of rats in each group;with the application of the chemotherapeutic drug oxaliplatin on the 6th day,the CINP chronic model The cold pain threshold of the rats in the group gradually decreased and continued until the 15 th day.The cold pain threshold was statistically different from the blank group at the time node(Day6,9,12,15)(P < 0.05),The western medicine duloxetine,Huangqi Guizhi Wuwu Decoction gavage and washing have a certain effect on the prevention and treatment of cold pain threshold drop.Western medicine duloxetine has a certain therapeutic effect on the prevention and treatment of oxaliplatin-induced cold pain threshold decrease.Compared with the model group,the cold pain threshold drop rate was low at(Day 6,9,12,15)(P < 0.05);Huangqi Guizhi Wuwu Decoction also has a certain therapeutic effect on the prevention and treatment of oxaliplatin-induced cold threshold decline.Compared with the chronic CINP model group,on(Day 6,12,15)The chilling pain threshold has a small decrease and has a statistical difference(P < 0.05);Huangqi Guizhi Wuwu Decoction can be used to effectively inhibit the cold pain threshold of oxaliplatin-induced chemotherapy in rats.There is no statistical difference(P > 0.05).Huangqi Guizhi Wuwu Decoction washing has a better effect on preventing and treating the oxaliplatin-induced cold pain threshold reduction.At the 12 th and15th days,the cooling threshold decreased by a small amount,with a statistical difference(P <0.05),compared with the western medicine duloxetine at(Day 12,15),and at the 15 th day Comparing the prevention and treatment of Huangqi Guizhi Wuwu Decoction by gavage,there was a statistical difference(P < 0.05),and the curative effect was outstanding.4.The results of the heat pain threshold of rats in each group showed that: in terms of the heat pain threshold of rats,there was no statistical difference in the heat pain threshold of rats in each group at baseline(the 0th day).Application,the chronic pain threshold of rats in the chronic CINP model group gradually decreased from the 3th day,and continued until the 15 th day.The thermal pain threshold of rats in the CINP chronic model group was(Day 3,6,9,12,15)There was a statistically significant difference between the time node and the blank group(P< 0.05).The western medicine duloxetine,Huangqi Guizhi Wuwu Decoction and gavage and washing have a certain effect on the prevention and treatment of heat pain threshold reduction.Western medicine duloxetine has a certain therapeutic effect on the prevention and treatment of oxaliplatin-induced decrease in heat pain threshold.Compared with the chronic CINP model,the decrease in heat pain threshold was low at(the 3th and 9th days),with statistical differences(P < 0.05);Huangqi Guizhi Wuwu Decoction has a certain effect on the prevention and treatment of heat pain threshold reduction.Compared with the CINP chronic model,at(the 12 th day)the decrease in heat pain threshold is low,with statistical differences(P < 0.05);Huangqi Guizhi Wuwu Decoction for external use has a good effect on the prevention and treatment of oxaliplatin-induced reduction of heat pain threshold in rats,at multiple time points(Day 9,12,15)When compared with the blank group,there was no statistical difference(P > 0.05),and the prevention and treatment effect was better than that of the duloxetine and Huangqi Guizhi Wuwu Decoction(P < 0.05).5.The pathological morphological observation results of neurons in each group of rats showed that the dorsal root ganglion cells in the spinal cord of the normal group were regularly arranged In:the blank group,the spinal dorsal root ganglion cells in the spinal cord were arranged regularly,with normal cell spacing and normal cell size;the arrangement of the spinal dorsal root ganglion cells in the chronic CINP model group was disordered The cell spacing is normally increased,the cell nucleus becomes smaller,and suspected apoptotic cells can be seen,which are quite different from normal nerve cells;the arrangement of spinal dorsal root ganglion cells in the western medicine group of rats is roughly regular,with local cell spacing increased and the cell nucleus slightly reduced;The dorsal root ganglion of spinal cord of Huangqi Guizhi Wuwu Decoction gavage group was roughly regular,with increased cell spacing,slightly reduced cell nucleus,and a small amount of apoptosis,which was not significantly different from the Western medicine group;Huangqi Guizhi Wuwu washing group of rat spinal dorsal root ganglion cells arranged regularly,with normal cell spacing and normal cell size,and no significant difference from normal nerve cells.6.The content of TNF-α,IL-1β and IL-6 inflammatory factors in the serum of rats in each group showed that:compared with the blank group,chronic CINP model group,western medicine duloxetine group,Huangqi Guizhi Wuwu Decoction gavage and washing group,the serum TNF-α content was significantly increased,the difference was statistically significant(P< 0.05);compared with the chronic CINP model group,the western medicine duloxetine group,Huangqi Guizhi Wuwu Decoction gavage group and Huangqi Guizhi Wuwu Decoction washing control group,the serum TNF-α content was significantly reduced,the difference was statistically significant(P < 0.05);compared with the western medicine duloxetine group,astragalus There was no statistically significant difference in the content of TNF-αbetween Guizhi Wuwu Decoction Gavage Prevention and Treatment Group and Huangqi Guizhi Wuwu Decoction washing Group(P > 0.05);Compared with the Huangqi Guizhi Wuwu Decoction gavage control group,the water content of TNF-α in the Huangqi Guizhi Wuwu Decoction washing group decreased,and the difference was statistically significant(P< 0.05).Compared with the blank group,the serum level of IL-1β in the chronic CINP model group,the western medicine duloxetine treatment group,the Huangqi Guizhi Wuwu Decoction gavage and washing group increased significantly,the difference was statistically significant(P < 0.05);compared with the chronic CINP model group,the western medicine duloxetine group,Huangqi Guizhi Wuwu Decoction gavage control group and Huangqi Guizhi Wuwu Decoction washing control group,serum IL-1β value decreased significantly,the difference was statistically significant(P < 0.05);compared with the western medicine duloxetine group,the amount of IL-1β in the Huangqi Guizhi Wuwu Decoction gavage control group and Huangqi Guizhi Wuwu Decoction foaming group,The difference was not statistically significant(P > 0.05);compared with Huangqi Guizhi Wuwu Decoction gavage control group,the content of IL-1β in Huangqi Guizhi Wuwu Decoction foaming group was decreased,the difference was statistically significant(P < 0.05).Compared with the blank group,in the chronic CINP model group,the western medicine duloxetine treatment group,the Huangqi Guizhi Wuwu Decoction gavage control group and the Huangqi Guizhi Wuwu Decoction washing group,the serum IL-6 content increased significantly,the difference Statistically significant(P < 0.05);compared with the chronic CINP model group,the serum IL-1β in the western medicine duloxetine group,Huangqi Guizhi Wuwu Decoction gavage control group and Huangqi Guizhi Wuwu Decoction foaming control group The content was significantly reduced and the difference was statistically significant(P < 0.05);compared with the western medicine duloxetine group,the content of IL-6 in the Huangqi Guizhi Wuwu Decoction gavage control group was increased,the difference was statistically significant(P <0.05),IL-6 content in Huangqi Guizhi Wuwu Decoction washing Group,the difference was not statistically significant(P > 0.05);compared with Huangqi Guizhi Wuwu Decoction gavage control group,IL-6 in Huangqi Guizhi Wuwu Decoction washing Group content decreased,the difference was statistically significant(P < 0.05).7.The results of WB detection of ERK1 / 2,p38,JNK,c-Fos,CREB and Nf-κB protein in L4-L5 spinal dorsal root ganglion tissues of rats in each group showed that:compared with the blank group,the CINP model group,the rat L4-L5 spinal cord after using oxaliplatin In dorsal root ganglion cells,ERK1/2,p38,JNK,c-Fos,CREB,Nf-κ B protein expression was significantly increased(P < 0.05);Western medicine group,Huangqi Guizhi Wuwu Decoction gavage and washing group all can significantly reduce the protein expression levels of ERK1/2,p38,JNK,c-Fos,CREB,Nf-κ B(P < 0.05).There was no significant difference in protein expression levels of JNK,CREB and Nf-κB in the western medicine group,Huangqi Guizhi Wuwu Decoction gavage and external bubble washing group(P > 0.05).Western medicine group and Huangqi Guizhi Wuwu Decoction external bubble washing group,There was no significant difference in the effect of reducing ERK1/2,p38 and c-Fos and protein expression levels(P > 0.05),but they were all better than Huangqi Guizhi Wuwu Decoction gavage group(P < 0.05).8.The results of IHC detection of ERK1 / 2,p38,JNKprotein in L4-L5 spinal dorsal root ganglion tissues of various groups of rats showed that:compared with the blank group,the CINP model group,the rat L4-L5 spinal cord after using oxaliplatin in dorsal root ganglion cells,the expressions of ERK1/2,p38,JNK,and protein were significantly increased(P <0.05);the Western medicine group,Huangqi Guizhi Wuwu Decoction gavage and washing group could significantly reduce ERK1/2,p38 and JNK protein expression level(P <0.05);Western medicine group,Huangqi Guizhi Wuwu Decoction gavage and external washing group ERK1/2,p38 and JNK protein expression levels were not significantly different(P > 0.05).Conclusion:1.Huangqi Guizhi Wuwu Decoction can improve the slow weight gain of oxaliplatin-induced chronic CINP model rats,effectively prevent and treat oxaliplatin-induced chronic CIPN rat model of hyperalgesia,and antagonize the mechanical pain threshold and cold pain Threshold and heat pain threshold decreased,and the effect of Huangqi Guizhi Wuwu Decoction external washing was better than that of Huangqi Guizhi Wuwu Decoction gavage.2.Huangqi Guizhi Wuwu Decoction can effectively antagonize the effect of oxaliplatin on the injury of spinal dorsal root ganglion cells in rats to achieve effective prevention and treatment,and the effect of external washing is better than that of Huangqi Guizhi Wuwu Decoction gavage.3.Huangqi Guizhi Wuwu Decoction can down-regulate the levels of TNF-α,IL-1β and IL-6inflammatory factors in the serum of chronic CINP rats induced by oxaliplatin,and reach the inhibition of MAPK signaling pathway family,which belongs to ERK1/2,p38,Activation of the JNK signaling pathway,thereby reducing the expression of downstream key proteins cFos,CREB and Nf-κ B,thereby achieving anti-neural cell apoptosis and reducing pain sensitization to prevent and repair the oxaliplatin-induced chemotherapy The role of neuronal injury in CINP rats. |
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