| Effect of therapeutic intraperitoneal injection of Iicochalcone A on neuropathic painObjectiveTo investigate the protective effect and evaluate the underlying mechanism of Lic-A against neuropathic pain in a rat model.Chronic constriction injury(CCI)surgery was employed in rats to establish neuropathic pain model.MethodsRats were intraperitoneally administrated with Lic-A daily from day 4 to day 10 after CCI surgery.The changes of PWT and PWL were measured on day 1 before CCI surgery and day 4,day 6,day 8,day 10 after CCI surgery.Previous experiments have observed stable mechanical and heat-induced pain on day 4 after CCI.In the initial experiment,we examined the effect of Lic-A on control/sham CCI rats.In the control/sham group,the sciatic nerve was only exposed without ligation.24 SD rats were randomized into 4 groups:Lic-A(1.25 mg/kg)group,Lic-A(2.50 mg/kg)group,Lic-A(5.00mg/kg)group,Vehicle group(constant volume DMSO+PBS).In the next experiment,90 SD rats were randomized into 5 groups(18 rats/group).They were control group,CCI group,L(L)group,L(M)group,L(H)group and Lic-A groups at three doses(1.25,2.50 and 5.00 mg/kg).Lic-A was dissolved in dimethylsulfoxide(DMSO)with the final concentration of DMSO less than 0.5%,and intraperitoneally administered after CCI surgery from the 4-th day to the 10-th day(7 days).The control group and CCI group were given the same volume of DMSO in phosphate-bufered saline(PBS).1.In the initial experiment and the next experiment,the PWT and PWL in rats at different time points of 1 day before surgery and the 4-th day,the 6-th day,the 8-th day and the 10-th day after surgery were measured.2.On the 10-th day after CCI,activation levels of microglia were detected.3.The proportion of M2-type microglia in each group was detected by immunofluorescence,and the expression level of M1-type microglia markers and M2-type microglia markers mRNA were determined by reverse transcription polymerase chain reaction(qPCR).4.The levels of IL-1?,TNF-?,IL-6 and IL-10 in spinal dorsal horn in different groups were measured by using ELISA kits.5.The levels of p38 and p-p38 in spinal dorsal horn of rats in each group were measured.Results1.Lic-A did not affect PWT and PWL of sham/control rats,but decreased PWT and PWL of CCI rats dose-dependently.Taken together,these results suggested that Lic-A could attenuate CCI-evoked neuropathic pain in rats.2.CCI induced significant microglia activation.Importantly,Lic-A treatment had no effect on the number of neurons,indicating that Lic-A showed no toxicity to neurons.IBA1 expression was also detected by Western blot analysis.CCI upregulated IBA1 expression.However,Lic-A treatment significantly inhibited IBA1 expression in CCI-treated rats with a dose-dependent manner.These results suggested that Lic-A could block CCI-induced microglia activation in rats.3.The CD206/IBA1 antibody was used for immunofluorescence double staining,which was consistent with the above experimental results.In the spinal dorsal horn of L4-6,the number of activated microglia in the control group was very small,while the number of activated microglia in the CCI group was significantly increased,and the number of activated microglia in the Lic-A group was less than that in the CCI group.The percentage of microglia co-stained by IBA1/CD206 antibody in L(H)group was higher than that in CCI group.QPCR was used to detect the mRNA levels of M1 and M2 microglia markers in the spinal dorsal horn of rats.After intraperitoneal injection of Lic-A,the mRNA levels of iNOS,CD32 and CD86 of M1 microglia in the spinal dorsal horn of rats were down-regulated,while the mRNA levels of CCL-22 and TGF-? of M2 microglia were up-regulated,which indicated that Lic-A could up regulate the proportion of M2 microglia in spinal dorsal horn of rats after CCI.4.Rats exposed to CCI showed notable release of IL-1?,TNF-? and IL-6.However,the release of IL-1?,TNF-? and IL-6 in CCI-treated rats was inhibited by Lic-A treatment with a dose-dependent manner.The release of IL-10 in CCI-treated rats was elevated by Lic-A treatment.These results demonstrated that Lic-A could inhibit CCI-induced release of inflammatory factors.5.CCI treatment enhanced p38 phosphorylation.However,Lic-A treatment dose-dependently inhibited p38 phosphorylation in CCI-treated rats.These results suggested that the inhibition of p38 phosphorylation contributed to Lic-A mediated protective effect against CCI evoked neuropathic pain in rats.ConclusionOur results indicated that Lic-A could attenuate CCI-evoked neuropathic pain in rats through inhibiting microglia activation,p38 phosphorylation and inflammatory response.Effect of prophylactic intraperitoneal injection of licochalcone A on neuropathic painObjectiveTo investigate the preventive effect of Lic-A on neuropathic pain in CCI rats and its dose-dependence and evaluate the underlying mechanism of Lic-A against neuropathic pain.The changes of PWT and PWL,neurons,glial cells,cytokines were measured by pretreating with Lic-A.MethodsIn the initial experiment,we examined the effect of Lic-A on control/sham CCI rats.24 SD rats were randomized into 4 groups:Lic-A(1.25 mg/kg)low-dose group,Lic-A(2.50 mg/kg)medium-dose group,Lic-A(5.00mg/kg)high-dose group,and Vehicle group(constant volume DMSO+PBS).In the next experiment,75 SD rats were randomized into 5 groups(15 rats/group).They were control group,CCI group,L(L)group,L(M)group,L(H)group,and Lic-A groups at three doses(1.25,2.50 and 5.00 mg/kg).Lic-A was dissolved in DMSO with the final concentration of DMSO less than 0.5%,and intraperitoneally administered after CCI surgery from the 3-th hour before CCI to the 10-th day after CCI(11 days).The control group and CCI group were given the same volume of DMSO in PBS.1.In the initial experiment and the next experiment,the PWT and PWL in rats at different time points of 1 day before surgery and the 1-th day,the 3-th day,the 7-th day,the 10-th day,the 14-th day and the 21-th day after surgery were measured.2.On the 10-th day after CCI,activation levels of microglia were detected.3.The level of IL-1?,TNF-?,IL-6 and IL-10 in spinal dorsal horn in different groups was measured.4.The levels of p38,p-p38,p65 and p-p65 in spinal dorsal horn of rats in each group were measured.Results1.Lic-A did not affect PWT and PWL of sham/control rats.Compared with Control group,the PWT of CCI group was significantly decreased at 1,3,7,10,14 and 21 days.Different doses of Lic-A inhibited the reduction of CCI-induced PWT to different degrees.The PWT of the Lic-A(H)group was significantly higher than the CCI group and maintained for at least 21 days after CCI,and the PWT of the Lic-A(M)group was maintained for at least 14 days.The PWT of the Lic-A(L)group was higher than that of the CCI group only on the 10th day after CCI.In this part of the study,we observed that a high dose of Lic-A(5.00mg/kg)prevented mechanically stimulated hyperalgesia in CCI rats for at least 11 days after intraperitoneal injection was discontinued.Moderate doses of Lic-A(2.50 mg/kg)last for at least 4 days,1.25 mg/kg Lic-A was not effective after discontinuation.The effect of Lic-A on PWL was similar to that of PWT.2.CCI induced significant microglia activation.Importantly,Lic-A treatment had no effect on the number of neurons,indicating that Lic-A showed no toxicity to neurons.IBA1 expression was also detected by Western blot analysis.CCI upregulated IBA1 expression.However,Lic-A treatment significantly inhibited IBA1 expression in CCI-treated rats with a dose-dependent manner.Taken together,these results suggested that Lic-A could block CCI-induced microglia activation in rats.3.Rats exposed to CCI showed notable release of IL-1?,TNF-? and IL-6.However,the release of IL-1?,TNF-? and IL-6 in CCI-treated rats was inhibited by Lic-A treatment with a dose-dependent manner.The release of IL-10 in CCI-treated rats was elevated by Lic-A treatment.These results demonstrated that Lic-A could inhibit CCI-induced release of inflammatory factors.4.CCI enhanced p38 and p65 phosphorylation.However,Lic-A treatment dose-dependently inhibited p38 and p65 phosphorylation in CCI-treated rats.These results suggested that the inhibition of p38 and p65 phosphorylation contributed to Lic-A mediated protective effect against CCI evoked neuropathic pain in rats.ConclusionThe injection of Lic-A at 3h before CCI inhibited the activation of microglia,and the release of pro-inflammatory mediators in spinal dorsal horn.The severity of neuropathic pain was effectively prevented.It is suggested that Lic-A may be a valuable plant extract for the prevention of neuropathic pain.Licochalcone A alleviates neuropathic pain in CCI rats through p38MAPK/NF-?B signaling pathwayObjectiveTo investigate whether Lic-A can inhibit the expression of inflammatory factors by inhibiting the activation of p38MAPK/NF-?B signaling pathway,and thus have analgesic effect on neuropathic pain.Methods30 SD rats were randomly divided into 5 groups:Control group,CCI group,CCI+Lic-A,CCI+SB203580 group and CCI+Lic-A+anisomycin group.The injection concentration of Lic-A was 5.00mg/kg.The time of intraperitoneal injection of Lic-A and intrathecal injection of SB203580 and anisomycin was the 3h before CCI to the 10-th day after CCI.The PWT and PWL of rats were measured before drug intervention and 1,3,7 and 10 days after CCI.At the 10-th day after CCI,samples were collected from each group and the levels of p-p38 and p-p65 were detected by Western Blot.The mRNA expression level of p-p65 was detected by qPCR.Results1.There was no significant difference in the basal value of PWT among Control group,CCI group and CCI+SB203580 group.At each time point of 1,3,7 and 10 days,the PWT of CCI group was significantly decreased compared with that of control group.The PWT of CCI+SB203580 group was higher than that in CCI group at 3,7 and 10 days.Intrathecal injection of SB203580 had a similar effect on PWL in rats.The levels of IL-1?,TNF-? and IL-6 in CCI+SB203580 group were lower than those in CCI group.The level of p-p65 in CCI+SB203580 group was lower than that in CCI group.2.The PWT of CCI group was significantly lower than that in control group since 1 day after CCI.The PWT of anisomycin+Lic-A group was significantly lower than that of Lic-A group at the same time points of 3d,7d and 10d.At the same time,intrathecal injection of anisomycin and intraperitoneal injection of Lic-A had similar effects on PWL.The expression of IL-1?,TNF-? and IL-6 was induced by CCI.At the same time,the levels of IL-1?,TNF-? and IL-6 in the anisomycin+Lic-A group were higher than those in the CCI+Lic-A group.The results showed that anisomycin could partially reverse the effect of Lic-A on the expression of IL-1?,TNF-? and IL-6 in the spinal dorsal horn of rats.ConclusionThe results of these studies showed that Lic-A could inhibit the activation of p38 MAPK/NF-?B signaling pathway,inhibit the release of inflammatory factors,and produce analgesic effects on neuropathic pain. |
PDF Full Text Request