The Regulatory Effect And Mechanism Of NLRP3 Signaling Pathway On Organ Injury

At present,the aging of population is an extremely serious social problem,which affects the development of society and economy.With the increasing aging problems of the society,the neurodegenerative diseases and various clinical symptoms such as respiratory failure,infection,shock and even acute lung injury and acute respiratory distress syndrome patients have increased dramatically.Studying the protective mechanisms of important organ injury,and developing possible regulatory targets and medicine have become urgent clinical problems.The innate immune system is the first defense line and plays an important role in the removal of foreign pathogens and development of adaptive immune responses.It is of great clinical significance to study protection and molecular mechanism of important organ injury in senile diseases.Innate immunity is the first defense line of body and plays an important role in removal of foreign pathogens and development of adaptive immune responses.NLRP3 inflammasome is closely related to senile diseases and biological injury,and it has great theoretical value and clinical significance to elucidate its intrinsic regulatory mechanism and development of possible regulatory medicine.Parkinson's disease(PD)is a typical neurodegenerative disease characterized by degeneration and loss of dopaminergic neurons.The main clinical manifestations are tremor,bradykinesia and stiffness.The main pathological features are progressive and extensive degeneration and loss of dopamine(DA)neurons in the substantia nigra pars compacta and Lewy body(LB)appears in cells.Hydroxysafflor yellow A(HYSA)is a glucoside compound extracted from the traditional Chinese medicine safflower(Carthamus tinctorius L).Studies have shown that HSYA has many therapeutic effect such as promoting circulation,neuroprotection,anti-oxidation and brain and myocardial ischemia-reperfusion injury,and its role in cardiovascular and neurodegenerative diseases has become a research hotspot.6-hydroxydopamine(6-OHDA)local striatal injection and 6-OHDA-induced human neuroblastoma cell(SH-SY5Y)injury model were widely used for medicine evaluation of PD.It has been reported that HSYA significantly improves 6-OHDA-induced dyskinesia and behavioral changes in PD mice,whereas little is known about regulation of NLRP3 inflammasome and MAPK pathways in 6-OHDA induced brain and neuron damage characterize by reduction of dopamine neurotransmitter.Studying its intrinsic molecular mechanisms is of great value in providing clinical potential therapeutic targets and possible medicine.In the first part of our study,6-OHDA-induced injury of dopaminergic neurons and SH-SY5Y cells were studied.As a traditional Chinese medicine safflower extract,the mechanism of HSYA was explored by detecting the effect of HSYA on NLRP3 and MAPK signaling pathways and it provided theoretical and experimental basis for development and utilization of medicine for brain injury protection.It was found that NLRP3 activation was closely related to dopaminergic neuronal injury in 6-OHDA-induced PD model,suggesting that dopaminergic neurons and NLRP3 inflammasome may regulate each other.Previous clinical studies have found that NLRP3 promotes inflammatory responses to induce dopamine neurons injury.Dopaminergic neurons can inhibit the activation of NLRP3,and it is indicated that the increase of dopaminergic neurotransmitters in the brain may regulate the activation of NLRP3 inflammasome.NLRP3 inflammasome plays an important role in progression of brain injury induced by 6-OHDA,and suggests that endogenous dopamine neurotransmitters play a role in the regulation of inflammasome.Dopamine(DA),as a neurotransmitter,not only regulates behavior,exercise,endocrine,cardiovascular,renal and gastrointestinal functions,but also is an important link between the nervous system and immune system.DA receptors exist in almost all immune cells and can regulate the proliferation of cells and production of cytokines.Previous studies have shown that alveolar macrophages(AMs)are the important target cells and main source of pulmonary cytokines in MV-induced lung injury.AMs account for about 5%of the peripheral cells of the alveolar,under physiological conditions.Mechanical stretch can activate AMs,producing a large number of chemotactic and cytokines,inflammatory factors recruiting concentrated granules,mononuclear cells,macrophages and lymphocytes,causing severe pulmonary inflammatory reactions and biological injury.Previous studies have focused on the regulation of inflammation and innate immunity in dopamine of central nervous system.However,the regulatory mechanisms of dopamine in NLRP3-mediated lung injury have not been fully elucidated.In the second part of study,mechanically stretched mice and cyclic stretch(CS)induced alveolar macrophages(AMs)were used to study the molecular mechanism of dopamine's regulation of NLRP3 inflammasome pathway,and to find effective treatment for lung biological injury.Part I:The Protective Effect of Hydroxysafflor Yellow A on 6-OHDA-induced Brain Injury through NLRP3 and MAPK PathwayObjective1.To study the protective effect and mechanism of hydroxysafflor yellow A(HSYA)on nigrostriatal dopaminergic neurons injury in PD mice model.2.To investigate the effect of HSYA on NLRP3 and MAPK signaling pathway protein expression in PD mice model.3.To investigate the protective effect of HSYA on 6-OHDA-induced inflammatory injury of SH-SY5Y cells.Methods1.Animal protocolPD model was prepared by injecting 6-OHDA into the right striatum under stereotaxic apparatus.C57BL/6J mice(n=80)were randomly divided into 5 groups:sham operation group(Sham),6-OHDA model group,6-OHDA model+HSYA(2 mg/kg)group,6-OHDA model group+HSYA(4 mg/kg),6-OHDA+HSYA(8 mg/kg)group,(n=16 per group).2.Behavioral testThe mice were intraperitoneally injected with HSYA or normal saline 15 minutes after the injection of 6-OHDA,and were treated once a day for two weeks.The control group was intraperitoneally injected with normal saline,and two weeks later,the Apomorphine(APO)-induced contralateral rotation was observed.3.High performance liquid chromatography for the determination of DA and metabolites(DOPAC and HVC)in brain.High performance liquid chromatography was used to detect the content of striatal dopamine(DA),dihydroxyphenylacetic acid(DOPAC)and high vanillic acid(HVA).The effects of HSYA on dopamine and metabolites in PD mice were investigated4.Dopaminergic neuron injury detectionImmunohistochemistry and western blot were used to detect the expression of the rate-limiting enzyme Tyrosine Hydroxylase(TH)and the number of dopaminergic neurons in the substantia nigra.5.Western blotBrain tissue was collected and western blotting was used to detect NLRP3 and MAPK signaling pathway-related proteins NLRP3,COX-2,iNOS,NF-?B p-p65,NF-?B p65,IKB?,p-ERK,ERK,p-JNK,JNK,p-p38,p38.6.Establishment of 6-OHDA cell injury modelCells were divided into 5 groups:Control group(Con);6-OHDA model group;6-OHDA+HSYA(1×10-6 mol/L)group;6-OHDA+HSYA(5×10-6mol/L)group;6-OHDA+HSYA(10-5 mol/L)Group.Human neuroblastoma SH-SY5Y cells were treated with normal saline or different concentrations of HSYA for 2 h,and exposed to 6-OHDA(50 ?M)for 24 h.7.CCK-8 was used to measure cell viability in SH-SY5Y cells.8.ELISA was used to detect IL-1? and IL-18 level in cell supernant.9.Western blot analysis of MAPK pathway related proteinCollecting cell proteins and detecting the levels of NLRP3,p-ERK,ERK,p-p38,p38,p-JNK and JNK proteins by western blot.Analysis of iNOS,COX-2 and NF-?B/I?B? protein expression levels in the presence of JNK phosphorylation agonists.Results1.HSYA improves 6-OHDA-induced contralateral rotation behavior in mice Compared with Sham group,the contralateral rotation induced by Apomorphine(APO)in 6-OHDA group was significantly increased(P<0.05);Compared with 6-OHDA group,the APO-induced contralateral rotation was significantly decreased in 6-OHDA+HSYA(8mg/kg)group(P<0.05).2.HSYA regulates 6-OHDA-induced striatum dopamine in mice The amount of striatal dopamine and its metabolites in 6-OHDA-induced mice was analyzed by liquid chromatography.The results showed that compared with the Sham group,dopamine in the striatum of the 6-OHDA group was significantly decreased(P<0.05).Compared with 6-OHDA group,dopamine in striatum of the 6-OHDA+HSYA(8mg/kg)group was significantly increased(P<0.05).3.HSYA protects 6-OHDA-induced brain dopaminergic neuronal injury The injury of substantia nigra pars copmacta(SNc)and striatal(Striatum,STR)dopaminergic neurons were evaluated by immunohistochemistry and detection of Tyrosine Hydroxylase(TH)protein.Immunohistochemical staining showed that TH-positive regions in SNc and STR were easily observed in Sham group.In 6-OHDA group,TH-positive neurons were significantly reduced(P<0.05).Compared to Sham group,6-OHDA resulted in a significant decrease in dopaminergic neuron levels in SN and STR(P<0.05),and HSYA(8 mg/kg)treatment significantly increased TH-positive neurons(P<0.05).The expression of TH protein was determined by western blot.The results showed that 6-OHDA significantly decreased TH protein expression compared with Sham group(P<0.05),while HSYA(8 mg/kg)intervention significantly increased TH protein expression in SN and STR(P<0.05).The above results indicate that striatal 6-OHDA injection induces significant injury of dopaminergic neurons,and HSYA has protective effects on injured dopaminergic neurons.4.HSYA regulates expression of NLRP3 protein level in brain.Western blot analysis showed the expression of NLRP3 in brain tissue of 6-OHDA group was significantly higher than that of Sham group,while HSYA dose-dependently decreased the expression of NLRP3 in brain tissue of 6-OHDA mice.5.HSYA regulates the expression of iNOS,COX-2 and NF-?B/I?B?Western blot results showed that the levels of iNOS,COX-2 and NF-?B p-p65 in brain tissue of 6-OHDA group were significantly higher than those in Sham group(P<0.05),while HSYA dose-dependently decreased the level of iNOS,COX-2 and NF-?B p-p65 induced by 6-OHDA injection.6.HSYA elevates SH-SY5Y cell viability The effect of 6-OHDA on cell viability in the presence of HSYA was detected by CCK-8.The results showed that the survival rate of 6-OHDA group was significantly lower than that of Con group(P<0.05),HSYA(5 ×10-6 and 10-)mol/L)significantly increased cell viability compared with 6-OHDA group(P<0.05).7.HSYA regulates MAPK pathway-associated protein expression In vivo,western blot results showed that compared with Sham group,6-OHDA significantly decreased p-ERK and increased p-JNK and p-p38(P<0.05).Compared with 6-OHDA group,HSYA dose-dependent increases the level of p-ERK in mice brain tissue and decreases the levels of p-JNK and p-p38.Western blot analysis showed that 6-OHDA+HSYA(10-5 mol/L)significantly increased p-ERK protein levels and decreased p-JNK and p-38 level in SH-SY5Y cells compared with 6-OHDA group(P<0.05).8.HSYA regulates the expression of iNOS,COX-2 and NF-?B/I?B?a and this effect is dependent on JNK phosphorylation.In vitro western blotting was used to detect iNOS,COX-2 and NF-?B/I?B?protein levels.Compared with 6-OHDA group,HSYA(10-5 mol/L)decreased iNOS,COX-2 and NF-?B p-p65 levels significantly P<0.05).The JNK phosphorylation agonist anisomycin partly reverses the HSYA's alleviation of 6-OHDA-induced inflammatory response,suggesting that HSYA's neuroprotection is dependent on JNK phosphorylation.Conclusion1.HSYA protects the dopaminergic neurons of PD mice model.2.HSYA regulates p38-MAPK and NLRP3 inflammasome pathway in 6-OHDA-induced PD mice model and SH-SY5Y cell injuries.Part II:The Protective Effect of Dopamine on Biological Lung Injury Induced by Mechanical Ventilation via the Inhibition of NLRP3 InflammasomeObjective1.To determine whether mechanical ventilation may induce cytokines,inflammatory mediators,and inflammatory cells to participate in biological lung injury.2.To explore whether dopamine may inhibit NLRP3 signaling pathway and elucidate the intrinsic molecular mechanism of protective effect of dopamine on lung injury.3.To determine whether cyclic stretch can induce alveolar macrophage cytokines,inflammatory mediators,and inflammatory cells to participate in biological injury.4.To elucidate whether dopamine can protect cyclic stretch induced alveolar macrophages injury by regulating NLRP3 signaling pathwayMethodsThis experiment is divided into two parts,in vivo and in vitro.In vivo(animal)experimental part1.1 The model of high tidal volume mechanical ventilation induced lung biological injury was established.The spontaneous respiration group was used as a control.The experiment was randomly divided into 5 groups:control group(Con);lung injury group(HV);lung injury+dopamine(10 mg/kg)group(HV+DA1);lung injury+dopamine(20 mg/kg)group(HV+DA2);lung injury+dopamine(30 mg/kg)group(HV+DA3).Mechanical ventilation was performed by an animal ventilator for 4 hours,with a tidal volume of 30ml/kg,respiratory rate of 50 times/min,peep of 0 cm H2O,I/E ratio of 1:2.The HV+DA group was intraperitoneally injected with different doses of dopamine,and the HV group was intraperitoneally injected with the same amount of normal saline.1.2 Bronchoalveolar lavage fluid(BALF)and lung tissue were collected to detect lung wet-to-dry ratio(W/D),myeloperoxidase(MPO),and alveolar lavage fluid protein level(pulmonary vascular permeability).1.3 Pathological changes of lung tissue were observed under light microscope,and semi-quantitative scores of lung tissue were observed.1.4 ELISA was used to detect the levels of TNF-?,1L-1?,IL-6 and IL-18 in BALF.1.5 RT-qPCR was used to detect mRNA expression levels of IL-1?,TNF-a,IL-6 and IL-18 cytokines in lung tissue.1.6 Western-blot was used to detect the effect of dopamine on the expression of NLRP3,Caspase-1 and IL-1? in lung tissue.2.In vitro(cell)experimental2.1 Study of the effect of dopamine on the activity of NLRP3 Alveolar macrophages were divided into the following group:control group(Con);cyclic stretch group(CS),cyclic stretch+dopamine(0.1 mM)group(CS+DA1);cyclic stretch+dopamine(0.2 mM)group(CS+DA2);cyclic stretch+dopamine(0.3 mM)group(CS+DA3).2.2 Alveolar macrophage culture and cyclic stretch2.2.1 Isolation of alveolar macrophages.The culture medium is RPMI-1640 medium containing 10%fetal bovine serum:the cell planting density is 1.0×105/cm~2,and the culture dish is pre-packaged with 0.1%collagen IV,and cultured in a 5%C02,95%air incubator(37?,saturated humidity).2.2.3 The collected cells were planted in a 6-well BioFlex baseplate,and the BioFlex culture dish was mounted on a FX-4000T Flexercell cell ring stretcher(Flexcell International,McKeesport,PA)25-mm BioFlex carrier,internal environment 5%CO2,95%air(37 C,saturated humidity).The computer sets the stretch parameter,the stretch frequency is 30 Cycles/min(0.5 Hz),and the stretch/relaxation ratio isl:1.The degree of distraction at the bottom of the culture dish was set to 20%,corresponding to 80%vital volume of mechanical ventilation,and the stretch time was set to 4h.2.3 Detection of various indicators2.3.1 RT-qPCR was used to detect the expression of IL-1? and IL-18 RNA.2.3.2 Western blot was used to detect the expression of NLRP3,Caspase-1 and IL-1?.Results1.Effects of dopamine on the pathological changes of lungs induced by mechanical ventilationTo study the protective effects of dopamine,pathological changes in lung tissue were assessed in mechanical ventilation(VIL1)lung injury.In mechanical ventilation with high tidal volume,compared with Con,the high tidal volume mechanical ventilation significantly caused alveolar edema and lung tissue damage(P<0.05).Dopamine significantly reduced edema and lung histopathological changes induced by HV.Dopamine(30 mg/kg,ip)significantly attenuated lung injury and histopathological scores(P<0.05).2.Dopamine significantly reduces the wet/dry ratio and myeloperoxidase(MPO)activity of lung tissueCompared with the Con group,the wet/dry(W/D)ratio of the HV group was increased.Dopamine(DA)treatment reduced the ratio of wet/dry(W/D)in a concentration-dependent manner.Compared with the Con group,MPO activity in HV group was significantly increased(P<0.05).MPO activity in the HV+DA3 group was significantly lower than that in the HV group(P<0.05).3.Dopamine reduces protein concentration in alveolar lavage fluid(BAL).Compared with the Con group,the HV group had an increased permeability of alveolar protein in the alveolar lavage fluid.Mechanical ventilation for 4 hours significantly increased the protein level in BAL.HV+DA1 and HV+DA2 significantly reduces the alveolar protein level compared with HV group(P<0.05).4.Dopamine reduces the levels of NLRP3 associated cytokines in alveolar lavage fluid(BAL)To use ELISA kit to measure the concentration of cytokines in the supernatant of alveolar lavage fluid(BAL).Compared with the Con group,high tidal volume mechanical ventilation significantly induced the production of IL-1?,TNF-a,IL-6 and IL-18 inflammatory factors(P<0.05).In addition,dopamine dose-dependently reduced proinflammatory cytokine levels in alveolar lavage fluid(BAL)in the lung injury group compared to the HV group.5.Dopamine reduces ventilator-induced inflammatory cytokine gene expressionReal-time quantitative PCR(RT-qPCR)was used to detect the expression of cytokines in lung tissue.The mRNA levels of IL-1?,TNF-a,IL-6 and IL-18 in the HV group were significantly higher than those in the Con group.However,the expression levels of IL-1?,TNF-?,IL-6 and IL-18 mRNA in HV+DA3 group were significantly lower than those in HV group(P<0.05).In vitro experiments,real-time quantification(qPCR)determines cytokine expression.The results show that cyclic stretch(CS)can increase IL-18,IL-1? expression.Dopamine can reduce the expression of IL-18 and IL-1? mRNA in macrophages in a dose-dependent manner.6.Dopamine inhibits the expression of NLRP3 inflammasome induced by mechanical ventilationThe production of IL-1? and IL-18 requires the presence and activation of NLRP3 inflammasome.In vivo experiments showed that high tidal volume mechanical ventilation significantly increased NLRP3 and caspase-1 protein expression(P<0.05).Dopamine dose-dependently inhibited its expression and decreased mechanical ventilation-induced IL-1? production.In vitro,Western Blot results showed that CS can increase the expression levels of NLRP3,Caspase-1,and IL-1?.The levels of NLRP3,Caspase-1,and IL-1? in CS group were significantly higher than those in the control group(P<0.05).Dopamine could inhibit the expression of NLRP3,Caspase-1 and IL-1? protein in a dose-dependent manner.ConclusionUnder the experimental conditions,the following conclusions can be drawn:1.Dopamine has a protective effect on biological lung damage caused by mechanical ventilation.2.Dopamine plays a protective role by regulating NLRP3 inflammasome signaling pathway and cytokine release to alleviate lung injury.3.Dopamine dose-dependently inhibits the activation of NLRP3 inflammasome signaling pathway in cyclic stretched alveolar macrophages and attenuates the expression of inflammatory cytokines.