| BackgroundNeuropathic pain(NP)is defined as a kind of pain caused by injury or disease acting on the somatosensory system.Clinically,it includes residual limb pain,phantom limb pain and post-stroke pain.Neuropathic pain is caused by high prevalence,complicated pathogenesis,difficult treatment,and sequelae,which causes a serious burden on the life and work of patients.The mechanism of NP includes two theories:Peripheral and central mechanisms,the conduction hub is in the dorsal horn of the spinal cord.In the early stage of NP,microglia in the spinal dorsal horn were activated and specific Iba1 was significantly expressed on the cell body surface.P38MAPK is widely distributed in activated microglia.Under pathological conditions,p38MAPK can mediate the activation of nuclear transcription factor-kappa B(NF-?B)at the transcription level,which enters the nucleus,combine with corresponding targets to promote the synthesis and release of inflammatory factors,such as IL-1??TNF-? And IL-6.The high expression of a large number of inflammatory factors not only amplifies the cascade effect of pain,but also further activates microglia.However,up to now,the drug treatment of neuropathic pain is still a thorny problem in the world.At present,there is an urgent need for new drugs with accurate therapeutic effect and less side effects.Current treatments for cellular molecules and targets in the spinal dorsal horn are hotspots.Studies this year have shown that traditional Chinese medicine bolaihui is effective for most chronic pain.It has abundant wild resources and is easy to cultivate.Sanguinarine(SG)is an alkaloid,SG has anti-inflammatory,protective nerve and myocardial,antibacterial,anti-osteoporosis,improvement of liver function,anti-tumor and insecticide and so on.With the development of molecular biology,the new efficacies of SG have also been continuously explored,which broadens the research field of SG,and provides a broad theoretical basis for SG's clinical application and basic research and development.Studies have shown that SG can inhibit Expression of IL-1??TNF-??IL-6;SG can also attenuate lipopolysaccharide(LPS)-induced interleukin(IL-?,IL-6)through the NF-?B signaling pathway,mRNA expression is up-regulated;SG can reduce the levels of TNF-? and IL-6,and the low expression of inflammatory factors and activated NF-?Bp65 are concentration-dependent;Recent studies have shown that SG inhibits the activation of MAPK and changes the synthesis and secretion of inflammatory mediators in vitro.In addition,SG can also significantly inhibit the activation of peritoneal mononuclear macrophages stimulated by LPS,and has a good protective effect on LPS-induced ulcerative colitis.However,there are basically no reports on the effect and mechanism of SG on NP.This study mainly is to explore the effects of SG on neuropathic pain and the possible molecular mechanisms involved in both in vivo and in vitro:Part ?Effects of sanguinarine on microglia and inflammatory factors in spinal dorsal horn of CCI ratsObjective:1.To study the effect of SG on rats in sham operation group(sham)by intraperitoneal injection of different concentrations of SG in sham operation model rats.2.Establish CCI animal model,observe the effect of SG on CCI animal model PWT and TWL,and further study the effect of SG on spinal dorsal horn microglia and inflammatory factors.Method:1.In order to study the effects of different concentrations of SG on rats,36 rats were randomly divided into 4 groups:sham operation group,sham operation+SG(1.00,2.5 and 6.25 mg/kg).Observe the pain behavior changes of rats at different time points before and after the operation on the 1,3,7,and 14 days.2.Adopt the CCI model.Microglia and Expression of TNF-??IL-1??IL-6 were detected by immunofluorescence and ELISA.Result:1.The effect of SG on mechanical pain threshold and thermal pain threshold of rats in sham group.Compared with the sham group(1.00,2.50,6.25 mg/kg)and the sham group,the PWT and TWL of rats basically had no change before and after operation.(P>0.05).The above results suggest that SG 1(.00,2.50,6.25 mg/kg)has no statistically significant effect on the pain threshold of sham-operated rats.2.The effect of SG on mechanical pain threshold and thermal pain threshold of CCI model rats.PWT and TWL remained unchanged in sham operation.PWT and TWL decreased at various time points after operation in the CCI group,with the largest decrease on the first day after the operation;compared with the CCI group,CCI+SG Group(1.00,2.50,6.25 mg/kg)PWT and TWL were significantly reversed on the 3rd,7th,and 14th day after operation.Among them,the CCI+SG group(6.25 mg/kg)had a more obvious effect.3.The effect of SG on IBA1+ cells in the spinal dorsal horn of CCI rats.First,we made a comparison within each group and found that the expression of IBA1+cells was less in the sham,and the number of IBA1+cells remained unchanged;In the CCI group,the number of IBA1+ cells in the dorsal horn of the spinal cord increased slightly,and significant activation of IBA1+ cells was found by cell count.The change was basically consistent with the pain threshold.The change trend of the CCI+SG group(1.00,2.50,6.25 mg/kg)was similar to that of the CCI group.Then we selected the 7th day and the 14th day where the changes were more obvious,and compared and analyzed between the groups and found that the number of IBA1+ cells in the CCI group increased.IBA1+ cells activation decreased in CC1+SG group(1.00,2.50 and 6.25 mg/kg).The dorsal horn of the spinal cord in the SG(6.25 mg/kg)group Among them,the expression of IBA1+ cells decreased the most.On the 14th day,the comparison between the groups was shown by the method of immunofluorescence merge.4.The effect of SG on TNF-?,IL-1? and IL-6 in the spinal dorsal horn of CCI rats.First,we made a comparison within each group.The expressions of TNF-?,IL-1? and IL-6 in the Sham group were basically the same before and after surgery.The expression of TNF-?,IL-1? and IL-6 in the CCI group increased after operation.In the CCI+SG group(1.00,2.50,6.25 mg/kg),compared with preoperatively,the expression of inflammatory factors in the spinal dorsal horn at each postoperative point showed an increasing trend.Then,the 7th and 14th days when the changes were more obvious were compared and analyzed between groups:The results of CCI group showed that the expression of inflammatory factors was up-regulated,CCI+SG group(1.00,2.50,6.25 mg/kg)reversed the above phenomenon.Conclusion:SG can increase PWT and TWL by down-regulating the activation of spinal dorsal horn microglia and the expression of inflammatory factors,thereby reducing NP.Part ?Effects of sanguinarine on p38MAPK/NF-KB signaling pathway in spinal dorsal horn of CCI ratsObjective:1.To explore the effect of SG on p38MAPK and NF-?B at the molecular protein level.2.To observe the activation of p38MAPK,NF-?Bp65 and the expression of inflammatory factors.Method:1.Experiment 1 was designed as follows:45 rats were randomly divided into 5 groups:sham group,CCI group,CCI+SG group(1.00,2.50,6.25 mg/kg).Through RT-PCR,WB,immunohistochemistry and other techniques,analyze the activation level of p38MAPK and NF-?Bp65 in the spinal dorsal horn by SG.2.We designed experiment 2 as follows:45 rats were randomly divided into 5 groups:sham group,CCI group,CCI+SB203580 group,CCI+SG(6.25 mg/kg)group,CCI+SG(6.25 mg/kg)+anisomycin group.Observe the pain behavior changes at different time points before and after the operation on the 1,3,5,and 7 days.The activation levels of inflammatory factors,p38MAPK and NF-?Bp65 in the spinal dorsal horn were analyzed by RT-PCR,WB,ELISA,immunehistochemistry and other techniques.Result:1.The effect of SG on the expression of p-p38 in spinal dorsal horn of CCI rats.The results showed that the expression of p-p38MAPK increased in the CCI group.However,intraperitoneal injection of SG can inhibit the up-regulation of p-p38 expression.2.Effect of SG on the activation of NF-?B p65 in the spinal dorsal horn of rats.Therefore,we used RT-PCR and WB technology to detect the activated NF-?Bp65 protein expression in the spinal dorsal horn of rats.Up-regulation of NF-?Bp65 expression in CCI group.Similarly,compared with the CCI group,SG(1.00,2.50,6.25 mg/kg)can significantly reduce the activation of NF-?Bp65 in the spinal cord of CCI model rats(P<0.05),and it shows a dose-dependent relationship,SG(concentration:6.25 mg/kg)has the best effect.3.Effects of SG on p38MAPK/NF-KB signaling pathway in spinal dorsal horn of rats.After proposing the above hypothesis,we carried out the following experiments to verify:1.SB203580 was injected into the subarachnoid space of CCI model rats.The results suggest that SB203580 increased PWT and TWL of CCI model rats.The intensity of action is slightly stronger than that of SG;we also found that SB203580 can significantly reverse the expression of NF-?Bp65 and can stimulate the secretion of TNF-?,IL-1? and IL-6,similar to the effect of the drug group;at the same time,our CCI model is large Rats were injected intrathecal with p38MAPK agonist anisomycin,and the results suggest that anisomycin can reverse the effect of SGConclusion:SG can down-regulate the expression of TNF-?,IL-? and IL-6 through the p38MAPK/NF-?B pathway.Part ?Effect of sanguinarine on LPS induced activation of BV-2 cells in vitroObjective:1.To study the effects of SG and LPS on the activation of BV-2 cells.2.Use a fluorescence microplate reader to detect the changes of ROS after SG on LPS stimulated BV-2,and use WB technology to-detect MAPKs,NF-?B,TNF-?,IL-6,IL-1? changes.and further detection of possible changes in JNK and ERK.Method:1.BV-2 cells were treated with LPS(20,40 ?g/ml)combined with SG(2.5?g/ml)for 48 hours,and cell viability was detected by MTT method.2.Continue the experiment with LPS of 10 ?g/ml.Treat BV-2 cells with LPS and SG for 48h,observe the morphology of BV-2 cells.3.LPS(10 ?g/ml)treats BV-2 cells for 0-48 hours,and fluorescence microplate reader detects the ROS production of BV-2 cells.4.LPS(10 ?g/ml)combined with SG(2.5 ?g/ml)was used to treat BV-2 cells for 48 hours,and he expression of MAPKs and NF-?B was detected by WB method.5.LPS(10 ?g/ml)combined with SG(2.5 ?g/ml)was used to treat BV-2 cells for 48 hours,and TNF-?,IL-6,and IL-1? were detected.Result:1.MTT method to detect the effect of SG and LPS on the activity of BV-2 cells.In the LPS(0-10?g/ml)group,there was no change in cell viability.The survival rate of cells in the LPS(20?g/ml)group decreased slightly.LPS(40 ?g/ml)treatment of BV-2 cells for 48 hours significantly inhibited cell growth and decreased cell viability by 18.4%.It was found that SG(0.1-2.5?g/ml)showed no obvious cytotoxicity within 48 hours.SG(5,10 ?g/ml)showed obvious cytotoxicity,and the cell survival rate was reduced by 26.8%and 53.9%,respectively(Figure 1B);when SG(2.5 ?g/ml)acts alone or with LPS(10 ?g/ml),the cell survival rate had no obvious change.The time-dependent results were further studied to confirm the cytotoxic effect of LPS(Figure 1C).However,when SG(2.5 ?g/ml)and LPS(20,40?g/ml)stimulated BV-2 cells at the same time for 48 hours.For example,as shown in Figure 1D,SG(2.5 ?g/ml)significantly improved cell viability from 82.7%to 100%.The above results show that:LPS(0-10 ?g/ml)and SG(0.1-2.5?g/ml)have no obvious toxic effects on BV-2 cells.When high concentrations of LPS(20,40 ?g/ml)stimulate BV-2 cells to produce toxicity,SG can inhibit LPS-induced BV-2 cytotoxicity at the cellular level.Choose LPS(10 ?g/ml)for subsequent experiments2.Cell morphology under the microscope.The Control group adhered very firmly,the cell body was irregularly elliptical,and the protrusions were thin and long.Compared with the Control group,after 48 hours of LPS stimulation,the cell body of BV-2 cells became larger and the cell protrusions increased and lengthened,indicating that BV-2 cells were activated;And SG can inhibit the activation of BV-2.3.SG inhibits the production of ROS induced by LPS.For example,as shown in 3A,LPS(10?g/ml)treatment of BV-2 cells can produce ROS in a time-dependent manner.The accumulation of ROS can be detected as early as 4h,and ROS increased relative to the control group at 24h.58.4%.However,co-treatment with SG(0.5,1,2.5?g/ml)can dose-dependently inhibit LPS-induced ROS production(Figure 3B).For example,SG(0.5,1,2.5 ?g/ml)reduced the level of ROS from 158%to 143.8%,135.3%,and 116.5%,respectively.Treatment of BV-2 cells with SG(2.5 ?g/ml)alone did not cause significant ROS production.The result of the ROS fluorescence photo in Figure 3C is the same as above.For example,the LPS(10 ?g/ml)group showed bright green fluorescence.After treatment with SG(0.5,1,2.5 ?g/ml),the fluorescence intensity decreased.The above results all show that:SG can reduce the generation of ROS.4.SG attenuates the expression of MAPKs and NF-?B in BV-2 cells induced by LPS.As shown in Figure 4,LPS(10?g/ml)treatment of BV-2 cells time-dependently activates the MAPKs pathway,which is manifested by the activation of Thr183-JNK,Thr180-p38,and Thr202-ERK proteins.We detected that LPS(10?g/ml)treatment of BV-2 cells activated Thr183-JNK and Thr202-ERK at the earliest 2h,and Thr180-p38 at the earliest 4h.Co-treatment with SG(2.5 ?g/ml)and LPS(10?g/ml)decreased the expression of Thr183-JNK,Thr180-p38,and Thr202-ERK;further studies showed that LPS(10 ?g/ml)treatment BV-2 cells activated the NF-?B pathway in a time-dependent manner.However,SG(2.5 ?g/ml)inhibited the activation of NF-?B.The above results all indicate that LPS(10 ?g/ml)treatment of BV-2 cells can cause the activation of MAPKs and NF-?B pathways.However,co-treatment with SG(2.5?g/ml)significantly inhibited the activation of MAPKs and NF-?B induced by LPS.5.SG attenuates the release of inflammatory factors in BV-2 cells induced by LPS.LPS(10?g/ml)increased the expression of IL-1?,TNF-?,and IL-6;however,the effect of LPS was reversed with SG.Therefore,we believe that SG can down-regulate the high expression of inflammatory factors.Conclusion:SG may reduce LPS-induced activation of BV-2 cells through the MAPKs/NF-?B pathway. |
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