Screening And Mechanisms Study Of Effective Parts Of Bauhinia Brachycarpa Benth On Neuropathic Pain

Objective: The effective part of ethnic medicine Bauhinia Brachycarpa Benth(BBB)on Neuropathic pain(NP)was screened based on the neuroinflammation via the methods of composition determination,analgesic model in vivo and inflammation model of macrophage in vitro.After that,the effect of this part on P2X4-BDNF-Trk B pathway in NP in vivo and in vitro was investigated.The results of this study will provide empirical evidence for the material basis and mechanism of BBB for the treatment of NP.The results of this study will also provide a theoretical basis for the folk medicine of BBB.Methods:1.Ethanol extract of Bauhinia brachycarpa Benth(EEBb)was prepared,and its extract portions were obtained with different organic reagents.These portions were petroleum ether extract(pe EEBb),ethyl acetate extract(ea EEBb)and n-butanol extract(nbEEBb).The contents of total flavonoids,total phenolic acids and total saponins in EEBb and the different extraction portions from EEBb were determined by aluminum nitrate complexation spectrophotometry,Prussian blue colorimetry and vanillal-perchloric acid colorimetry,respectively.2.Screen of the effective parts of Bauhinia Brachycarpa Benth on NP.(1)In vivo study.A mouse model of partial sciatic nerve ligation injury(PSNL)was established.The paw withdrawal mechanical threshold(PWMT)was determined,and the m RNA and protein levels of Iba-1(labeled activated microglia),CD16(labeled M1 microglia),CD206(labeled M2microglia)were detected by RT-q PCR and Western Blot.These results were used to evaluate the effects of EEBb and its different extraction portions on NP and the activation and phenotypic differentiation of microglia in NP.(2)In vitro study.Macrophage RAW264.7 was cultured in vitro and stimulated by lipopolysaccharide(LPS)or interleukin-4(IL-4)to differentiate macrophage into pro-inflammatory M1 type or anti-inflammatory M2 type,respectively.After drug treatment,the levels of nitric oxide(NO),inducible nitric oxide synthase(i NOS),tumor necrosis factor α(TNF-α)and interleukin-6(IL-6)in the supernatant were determined by ELISA.The m RNA expression of argininase-1(Arg-1)was detected by RT-q PCR.These results were used to evaluate the effects of EEBb and its different extraction portions on the function of different subtypes of macrophage.Finally,combined with the results of composition analysis,in vivo and in vitro experiments,the effective portion of BBB on NP were selected.3.Study on the mechanism of nbEEBb(the effective portion of Bauhinia Brachycarpa Benth)on NP.The potential targets and related pathways of BBB on NP were predicted by network pharmacology.PSNL model in vivo and microglia in vitro were used to investigate the effect of nbEEBb on the P2X4-BDNF-Trk B pathway,which is the neurotrophic factor signaling pathway that is closely related to the interaction of neurons with microglia.Specifically:(1)In vivo study.PSNL model in mouse was established and followed with the treatment of nbEEBb.The BDNF level in serum was detected by ELISA,and the m RNA expression of P2X4,BDNF and Trk B in tissues was detected by RT-q PCR.Western Blot was used to detect the protein level of P2X4,BDNF,Trk B,phosphorylation Trk B(p-Trk B),p38 MAPK and phosphorylated p38 MAPK(p-p38 MAPK)in tissue.(2)In vitro study.BV2 cells were cultured.Adenine nucleoside triphosphate(ATP)was used to activate purine receptors in the cell.After that,nbEEBb was added for treatment.The content of BDNF in supernatant was determined by ELISA,and the m RNA and protein levels of P2X4 and BDNF in cells were detected by RT-q PCR and Western Blot,respectively.And the protein levels of p38 MAPK and p-p38 MAPK were also measured by Western Blot.Results:1.The contents of total flavonoids and total phenolic acids in EEBb and its different extraction portions were nbEEBb > ea EEBb > EEBb > pe EEBb.The contents of total saponins were pe EEBb > nbEEBb > ea EEBb > EEBb.Therefore,nbEEBb contains the highest amount of active ingredients against NP.2.Screen of the effective parts of Bauhinia Brachycarpa Benth on NP.EEBb and its extracts could increase the PWMT of PSNL,restrained the activation of microglia,inhibited the pro-inflammatory M1 type of microglia,and promoted the anti-inflammatory M2 type of microglia.EEBb and its extracts also inhibited the secretion of NO,i NOS,TNF-α and IL-6by pro-inflammatory M1 type of macrophages,and increased the m RNA expression of Arg-1in M2-type macrophages.Thus,nbEEBb had the strongest ability to increase PWMT,reduce activation of microglia,inhibit M1-type of microglia,promote M2-type of microglia.At the same time,nbEEBb could significantly inhibit the secretion of pro-inflammatory factors in M1-type macrophages and promote the expression of anti-inflammatory factors in M2-type macrophages.Therefore,nbEEBb was the most effective part of EEBb against NP.3.Study on the mechanism of nbEEBb on NP.(1)The results of network pharmacology predicted that the core target of Bauhinia Brachycarpa Benth on NP was RAC-αserine/threonine protein kinase(Akt1),and the key pathway was the neurotrophic factor signaling pathway.(2)In vivo study.nbEEBb could significantly reduce the content of BDNF in serum,inhibit the m RNA and protein levels of P2X4,BDNF and Trk B in spinal cord tissue,and also inhibit the protein expressions of p-p38 MAPK and p-Trk B.(3)In vitro study.After the pathway was actived by ATP,nbEEBb could significantly reduce the content of BDNF in the supernatant,inhibit the m RNA and protein levels of P2X4 and BDNF,and decrease the protein levels of p-p38 MAPK.Conclusion: Among EEBb and its extracts,nbEEBb had the best effect on NP,and its mechanism was closely related to the inhibition of P2X4-BDNF-Trk B signaling pathway in neuron-microglia interactions.