| Background:Human immunodeficiency virus infection often causes nervous system diseases,which can be life-threatening in severe cases.HIV-1 envelope glycoprotein-120(gp120)mainly binds the viral envelope with the corresponding cell surface receptors of host cells,including CD4,B7 molecule and chemokine receptor complexes of T lymphocytes.Macrophages and microglia produce inflammatory cytokines and other neurotoxic toxins such as peanut-like acids to damage neurons and interfere with neurotransmitter functions.HIV-infected nerves cause pathological changes in the peripheral nerves,while the primary lesions may be in dorsal root ganglion(DRG).The results show that the gp120 can directly stimulate primary afferent neurons and cause pain allergy.The incidence of HIV infection associated with neuropathic pain is higher,and its mechanism is not clear,treatment is extremely difficult.The P2X4 receptor of DRG satellite glial cells and the P2X3 receptors of DRG neuron cells are closely related to neuropathic pain,and involve inflammation and immune reaction.Therefore,P2X4 and P2X3 receptors may be involved in gp120-associated neuropathic pain.Curcumin isolated from herb Curcuma rhizome has anti-inflammatory and anti-tumor effects.The water solubility,targeting and bioavailability of curcumin can be improved by nanoparticle encapsulation.At present,the effect of nanoparticle-encapsulated curcumin on P2X3 receptor-mediated HIV-gp120-associated neuropathic pain is still not studied.Traditional methods of treating pain are mainly directed at neurons,but studies have shown that glial cells(microglia and astrocytes)in the central nervous system can significantly affect the pathological changes of chronic pain(such as regulating the activity of spinal neurons).Therefore,most of the current failure of pathological pain treatment may be due to its targeting in neurons.Recent studies have shown that glial cells may be a more appropriate target for chronic pain prevention.The injured cells,stress cells and sensory nerve endings release a large number of ATP.ATP plays an important role in the signaling transmission of pain and its related injury.P2X4 receptor in satellite glial cells can trigger inflammation in response to high ATP release.At present,it is not clear that the whether P2X4 receptor in the satellite glial cells of dorsal root ganglia is involved in the mechanism of gp120 mediated pyroptosis?Part one: Effects of nanoparticle-encapsulated curcumin on HIV-gp120-associated neuropathic pain induced by the P2X3 receptor in dorsal root gangliaObjective: In this study,a HIV gp120-induced neuropathic animal model was established.At the animal level,the study was aimed to investigate the effects of nanoparticle-encapsulated curcumin on the rat DRG P2X3 receptors-mediated HIV-1gp120-associated neuropathic pain and its possible mechanism.Methods: In this study,sciatic nerve injury model induced by gp120 will be used to explore the mechanism of gp120 related neuropathic pain and the effects of curcumin nanoparticles on P2X3 receptor mediated HIV-related neuropathology.There were two parts in the experiments of animall level.The experiments in the first part were divided into: normal group(control),sham operation group(sham),gp120 model group(gp120),gp120 combined with nano carrier group(gp120 + nano carrier),gp120 combined with nano carrier curcumin group(gp120 + nano curcumin).The experiments in the second part were divided into: normal group(control),gp120 model group(gp120),gp120 combined with nano carrier curcumin group(gp120 +nano curcumin),gp120 combined with P2X3 antagonist specific antagonist A-317491(gp120 + A-317491).Contents of the experiment were as follows:(1)Behavioral testing was applied to mesure mechanical withdrawal threshold(MTW)and thermal withdrawal latency(TWL)in the rats.(2)Real-time PCR method was used to detect the expression of P2X3 receptor m RNA in the DRG.(3)Western blotting was used to detect the protein expression changes of P2X3 receptor,ERK1/2,phospholated-ERK1/2(p-ERK1/2).(4)Immunohistochemical method was used to detect the expression of P2X3 receptor in the DRG neurons.(5)Electrophysiological was used to record the expression of P2X3 receptor agonist activated current in DRG neurons.(6)Bioinformatics was used to detection of Molecular docking between curcumin and human P2X3(h P2X3)protein.Results:(1)Behavioral results showed that: At 7 to 14 days after the operation,the MWT and TWL in the gp120 group were lower than that in the control group(p<0.01).There was no difference in the MWT and TWL between the gp120 + nano carrier group and the gp120 group(p>0.05).The MWT and TWL in the gp120 + nano curcumin group was higher than those in the gp120 group from days 7 to 14(p<0.01).(2)The expression level of P2X3 m RNA in the DRG of gp120 group was significantly higher than that in control group(p<0.01).No significant difference was found between gp120 + nano carrier group and gp120 group(p>0.05).The expression level of P2X3 m RNA in the gp120 + nano curcumin group was lower than that in the gp120 group(p <0.01).(3)The expression levels of P2X3、p-ERK protein in the DRG of gp120 group was significantly higher than that in control group,respectively(p<0.01).No significant difference was found between gp120 + nano carrier group and gp120 group(p >0.05).The expression levels of P2X3,p-ERK,in the gp120 + nano curcumin group was significantly lower than that in gp120 group,respectively(p<0.01).(4)The expression level of the P2X3 immunoreactivity in the DRG of gp120 group was significantly higher than that in control group(p<0.01).No significant difference was found between gp120 + nano carrier group and gp120group(p>0.05).The expression level of the P2X3 immunoreactivity in the gp120 +nano curcumin group was significantly lower than that in gp120 group(p<0.01).(5)The typical current trace induced by P2X3 agonist α,βme-ATP.α,βme-ATP(10μM)-activated currents in DRG neurons cultured with gp120 were higher than those in the controls.P2X3 agonist α,βme-ATP-activated currents in DRG neurons co-treated with gp120 plus nano curcumin were significantly inhibited compared with those from the DRG neurons that were only cultured with gp120.P2X3 antagonist A-317491 inhibited α,βme-ATP-induced currents in the DRG neurons cultured with gp120(p<0.01).(6)Molecular docking of curcumin on a h P2X3 protein were generated by the Auto Dock 4.2.Docking score of h P2X3 and curcumin(-7.7,kcal/mol)showed that curcumin was enabled the perfect fit to interact with h P2X3 receptor.The perfect match enabled the curcumin to interact with residues in the ATP-binding site.Part two: Protective role of dihydrolycorine against gp120-induced pyroptosis involved in the upregulated P2X4 receptor of satellite glial cells of dorsal root gangliaObjective: In this study,a cell damage model for DRG satellite glial cells(SGCs)treated with gp120 was established.At the cellular level,we studied whether P2X4 receptor was involved in HIV-gp120 induced satellite glial cell damage,and observed the effect and possible mechanism of dihydroalliine on HIV-gp120 related satellite glial cell injury mediated by P2X4 receptor.This study will provide a new way for prevention and treatment of HIV related neuropathic pain.Methods: In this study,SGCSdamage model was established by gp120 treatment.The role of P2X4 receptor in cell damage will be studied.There were seven parts in the experiments of cell level.The experiments in the first part were divided into normal group(control),HIV-1 coated glycoprotein gp120 group(200 pmol/L,400pmol/L,800 pmol/L,1600 pmo/L).The experiments in the second part were divided into the normal group(control),dihydrolycorine treatment group(1 μM/L,10 μM/L,100 μM/L,1000 μM/L).The experiments in the third part were divided into the normal group(control),dihydrolycorine treatment group(1μM/L,20 μM/L,40 μM/L,80 μM/L,160 μM/L).The experiments in the fourth part were divided into the normal group(control),HIV-1 envelope glycoprotein gp120 group(gp120),HIV-1glycoprotein gp120 plus dihydrolycorine treatment group(gp120 + DL).The experiments in the fifth part were divided into the normal group(control),normal group plus negative control sh RNA treatment group(control + NC sh RNA),normal group plus P2X4 sh RNA-1 treatment group(control + P2X4 sh RNA-1),the normal group plus P2X4 sh RNA-2 treatment group(control + P2X4 sh RNA-2),normal group plus P2X4 sh RNA-3 treatment group(control + P2X4 sh RNA-3).The experiments in the sixth parts were divided into normal group(control),HIV-1 envelope glycoprotein gp120 group(gp120),HIV-1 coated glycoprotein,gp120 plus ATP treatment group(gp120 + ATP).The experiments in the seventh parts were divided into the normal group(control),HIV-1 glycoprotein gp120(gp120)group,HIV-1 gp120 plus P2X4 sh RNA treatment group(gp120 + P2X4 sh RNA)and HIV-1 glycoprotein gp120 plus negative control sh RNA treatment group(gp120 + NC sh RNA),HIV-1 glycoprotein gp120 plus dihydrolycorine treatment group(gp120 + DL).The experiments included as following:(1)MTS,Enzymatic or TUNEL methods were used to detect the cultured SGC damage or pyroptosis.(2)Real-time PCR method was used to detect the expression of P2X4,interleukin 1 beta(IL-1β),Caspase-1,IL-18 and NLRP1 m RNA in SGCs.(3)Western blotting was used to detect the expression of P2X4 receptor,p38,phospholated-p38(P-p38),Caspase-1,NLRP1,inflammatory factor IL-1β,IL-18 protein in SGCs.(4)Enzyme linked immunosorbent assay(ELISA)was used to detect the release of tumor necrosis factor alpha(TNF-α),IL-1 β and IL-18 in the supernatant of in SGCs.(5)The content of NO,the amount of ATP release and the level of ROS in the supernatant of the cultured SGCs were detected by using the testing kits.Results:(1)Screening of gp120 concentration in the experiments: Screening results in MTS showed that,compared with the Control group,the cell survival rate in the concentration of 200 pmol/L gp120 treatment group was above 90%(p<0.05);the cell survival rate in 400 pmol/L group was reduced to 80%(p<0.01);the cell survival rate in 800 pmol/L group reduced to 57%(p<0.01);the cell survival rate in 1600 pmol/L group reduced to 47%(p<0.01).The expressions of P2X4 m RNA and protein in primary cultured SGCs were measured from 200 to 1600 pmol/L for gp120 concentration.Testing results showed that the expression levels of P2X4 m RNA and protein in the SGCs were significantly increased when the concentration of 400pmol/L gp120.Therefore,400 pmol/L was selected as the optimal concentration for cell injury model.(2)Screening of dihydrolycorine concentration in the experiments:MTS showed that,no difference was found between the dihydrolycorine group(at1μM/L concentration)and the control group(p>0.05).The survival rates of the dihydrolycorine group(at 1 μM/L,10 μM/L and 100 μM/L concentrations)were about 95%-75%(p<0.05),and the number of cell deaths in treatment group of 1000μM/L is very large.We further identified the effects of 5 concentration gradients(1μM/L,20 μM/L,40 μM/L,80 μM/L,160 μM/L)on 400 pmol/L gp120-induced cell injury model were observed.This result showed that the cell viability at 40 μM/L was less decreased compared with control(p<0.05);the cell viability at 80 μM/L was further decreased compared with control(p<0.01);and the significant cell cytotoxicity at 160 μM/L began to appear.Therefore,the concentration at 80 μM/L was chosen as the suitable concentration of dihydrolycorine.(3)In 400 pmol/L gp120-induced cell injury model,MTS showed that,the cell survival rate in the gp120 plus dihydrolycorine group was higher than that in the untreated gp120 group(p<0.01).Present data indicated that dihydrolycorine may improve the cell damage induced by gp120.(4)The cell morphology for primary cultured satellite glial cells was observed under inverted microscope.Spindle shaped,long dendrites,obvious axon dendrites were showed in control satellite glial cells.Cell swelling,nucleus concentration,and axonal tree short were displayed after the satellite glial cells treated with 400 pmol/L gp120 for 24 h.In gp120 plus dihydrolycorine(80 μM/L)treatment group,the cell status was significantly improved.(5)Screening of P2X4 sh RNA sequences was made to decide the sequence of best interference efficiency.Real-time PCR test results showed that the interference efficiency of the first pairs in three P2X4 receptor sh RNA sequences was the best than other in the same time.(6)The results of excellular ATP(e ATP)test in the supernatant of primary cultured SGCs indicated: the content of e ATP in the gp120 group was significantly increased compared with control group(p<0.01).(7)The expression levels of P2X4,NLRP1,Caspase-1,IL-1β,IL-18 m RNA in primary cultured SGCs were assayed: the expression levels of P2X4,NLRP1,Caspase-1,IL-1β,IL-18 m RNA in the gp120 group were significantly increased compared with control group,respectively(p<0.01).The expression levels of P2X4,NLRP1,Caspase-1,IL-1β,IL-18 m RNA in the gp120 + P2X4 sh RNA and gp120 + DL were significantly lower than those in untreated gp120 group,respectively(p<0.01).There was no significant difference between gp120+NC sh RNA group and the gp120 group(p>0.05).(8)The expression levels of P2X4,NLRP1,Caspase-1,IL-1β,IL-18,p38,P-p38 protein in primary cultured SGCs were tested: the expression levels of P2X4,NLRP1,Caspase-1,IL-1β,IL-18,P-p38 protein in the gp120 group were significantly increased compared with Control group,respectively(p<0.01).The expression levels of P2X4,NLRP1,Caspase-1,IL-1β,IL-18,P-p38 protein in the gp120 + P2X4 sh RNA and gp120 + DL were were significantly decreased compared with in the untreated gp120 group(p<0.01).There was no significant difference between gp120+NC sh RNA group and the gp120 group(p>0.05).(9)The results of LDH in primary cultured SGCs were detected: compared with Control group,the level of LDH in gp120 group has significantly increased(p<0.01).The level of LDH in the gp120 + P2X4 sh RNA and gp120 + DL were decreased compared with the untreated gp120 group(p<0.01).There was no significant difference between gp120+NC sh RNA group and the gp120group(p>0.05).(10)The results of peroxidase and inflammatory factors in the primary cultured SGCs were measured,the content of NO,TNF-α,IL-1β and IL-18 in the supernatant and the level of intracellular ROS in primary cultured SGCs were significantly increased in gp120 group compared with Control group,respectively(p<0.01).the content of NO,TNF-α,IL-1β and IL-18 in the supernatant and the level of intracellular ROS in the gp120 + P2X4 sh RNA and gp120 + DL were were significantly decreased compared with the untreated gp120 group(p<0.01).There was no significant difference between gp120+NC sh RNA group and the gp120 group(p>0.05).(11)The cell pyroptosis rate in the primary cultured SGCs was assayed: in gp120 group,pyroptosis in primary cultured SGCs was significantly increased compared with Control group,respectively(p<0.01).The pyroptosis rate in the gp120 + P2X4 sh RNA and gp120 + DL were improved compared with the untreated gp120 group(p<0.01).No significantly difference was found between the gp120 +NC sh RNA group and the gp120 group(p>0.05).Conclusion: The results in animal experiments showed that gp120 induced the up-regulation of DRG P2X3 receptor in neuropathic pain rats accompanied with up-regulation of p-ERK,mechanical hyperalgesia and thermal hyperalgesia.Curcumin nanoparticles can alleviate the P2X3 receptor mediated gp120 related neuropathic pain.The data in cell experiments revealed that HIV-1 envelope glycoprotein gp120 resulted in the elevated P2X4 receptor in the SGCs and incited the SGC injury / pyroptosis.The up-regulation of SGC P2X4 receptor activated pyroptosis induced by gp120 through NLRP1-mediated caspase-1 signaling.Dihydrolycorine treatment improved P2X4 receptor activated pyroptosis induced by gp120 through NLRP1-mediated caspase-1 signaling. |
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