The Mechanism Of TLR4/TAK1/IRF7 Axis Regulating Pyroptosis Through NLRP3 In Parkinson’s Disease

Background:Parkinson’s disease(PD)is the second most common neurodegenerative disease.At present,there is no radical cure,which causes a heavy burden to society and families.Therefore,it is urgent to explore the pathogenesis of PD in depth to provide a theoretical basis for the development of new therapeutic drugs.Although the typical cytopathologic features of PD are known to be the formation of lewy bodies in the cytoplasm of neurons and the progressive death of dopaminergic neurons,the specific mechanism of death remains unclear.As a new inflammatory programmed death mode,pyroptosis is attracting wide attention from researchers.Pyroptosis refers to the activation of inflammatory bodies,such as NOD-like receptor protein 1(NLRP1),NOD-like receptor protein 3(NLRP3)and NLR family CARD domain-containing protein 4(NLRC4),then they activated caspase-1 to promote the cleavage of gasdermin D(GSDMD)and the mature release of interleukin18(IL-18)and interleukin-1β(IL-1β),leading to the formation of membrane pores and the process of cell rupture and death.The NLRP3 inflammasome,as a key protein mediating pyroptosis,plays a bridging role in this process.Although current studies suggest that activation of NLRP3 inflammasome can induce pyroptosis of DA potent nerve cells and promote PD pathogenesis,the specific mechanism remains to be clarified.Previous work of our research showed that the expression of toll-like receptor 4(TLR4),NLRP3,IL-1β,tumor necrosis factorα(TNF-α)and other inflammation-related molecules were significantly up-regulated in PD rat models.In order to explore the mechanism,further RNA sequencing analysis revealed the activation of genes and signaling pathways related to innate immunity,including TLR4 signaling pathway,the initial link of innate immune response,and interferon regulatory factor 7(IRF7),a key transcription factor of innate immune response,suggesting that both may be involved in the pathogenesis of PD.Currently,TLR4 has been shown to activate IRF7 by regulating the expression of Transforming growth factor-β-activated kinase 1(TAK1),and IRF7 is involved in pyroptosis.However,it remains to be explored whether IRF7 in PD is also regulated by this pathway,what is the correlation between IRF7 and NLRP3 inflammatorome,and how they play a role in neuronal pyroptosis.To clarify the above problems,we propose the following scientific hypotheses:When TLR4 is activated by damage-associated molecular patterns(DAMPs)or pathogen-associated molecular patterns(PAMPs),IRF7 is upregulated by regulating the expression of downstream molecule TAK1,and IRF7 is then targeted to regulate the expression of NLRP3 to affect the pyroptosis of nerve cells,thus participating in the pathogenesis of PD.The elucidation of this mechanism suggests that innate immune response is involved in the pathogenesis of PD,which is expected to provide a new theoretical basis and therapeutic target for PD treatment.Purposes:It was verified that NLRP3 can promote PD by mediating neuronal pyroptosis.To explore the regulatory effect of TLR4/TAK1 signaling pathway on IRF7.To elucidate the molecular mechanism of NLRP3 regulation by IRF7 on neuronal pyroptosis.Methods:1.The PD rat model was established by intracerebral stereodirectional injection of 1-methyl-4-phenyl pyridinium(MPP~+),and the behavioral changes of the rats were observed by forelimb placing test,open field test and rotarod test.Real-time quantitative PCR(RT-PCR),Western blotting(WB),immunohistochemistry(IHC)and immunofluorescence(IF)were used to detect the molecular markers of apoptosis,pyroptosis,autophagy,inflammation and oxidative stress in PD model group and sham operation group.Subsequently,the rat adrenal pheochromocytoma cells(PC12)were treated with MPP~+to construct the PD cell model,and the changes of pyroptosis level in the PD cell model were detected by RT-PCR,WB,flow cytometry,CCK-8,lactate dehydrogenase(LDH)release assay and enzyme-linked immunosorbent assay(ELISA).2.RNA sequencing(RNA-seq)analysis was conducted on the brain tissues of PD rat models and rats in the sham operation group to excavate the differential genes in the samples of the two groups;Subsequently,m RNA and protein expression levels of differential genes were verified in PD rat model and PD cell model by RT-PCR and WB.3.The IRF7 overexpression cell model or IRF7 gene silencing cell model was constructed by lentivirus infection or transfection of si RNA interference fragments.After the successful construction of the cell model,the cell viability,LDH release,NLRP3,pyroptosis related genes and the expression changes of PD pathological markers tyrosine hydroxylase(TH)were detected in the model.Double luciferase assay was used to detect the binding between IRF7 and the NLRP3 promoter.It was confirmed that IRF7 affects nerve cell’s pyroptosis by targeting NLRP3 expression.4.Through in-depth mining of rat brain RNA sequencing data,the signaling pathway with significantly enriched differentially expressed genes was found in the two groups of samples,and the expression of key molecules in the signaling pathway was verified by RT-PCR and WB;By adding TAK1 pathway inhibitor(5Z)-7-Oxozeaenol to the PD cell model,a pathway activation or blocking cell model was constructed,and the changes in the expression of IRF7 and NLRP3 related molecules of this signaling pathway were detected in the model,so as to explore the molecular mechanism of the effect of TAK1 pathway on the expression of NLRP3 by regulating IRF7.To determine the effect of TAK1/IRF7 signaling pathway through NLRP3 on neurons pyroptosis,we detected the changes of pyroptosis level and TH expression in the cell model.Finally,by adding(5Z)-7-Oxozeaenol to the IRF7overexpressed cell model,the expression of NLRP3 and TH and the change of pyroptosis level in the cell model were detected,further confirming that TLR4/TAK1regulates NLRP3 through IRF7,thus affecting the pyroptosis of nerve cells and promoting the onset of PD.Results:1.Increased pyroptosis level in PD model(1)Pyroptosis increased in PD rat models:We first successfully constructed a rat model of PD by intracerebral stereotactic injection of MPP~+.The results of animal behavior showed that the forelimb placement error rate of PD model group was higher than that of sham operation group.Open field test showed that the total walking distance of PD model group was shortened and the time of staying in the central area was shorter than that of sham operation group.The results of rotarod test showed that PD model group had shorter stay time on the rod compared with sham operation group.These results suggest that the motor ability and limb coordination of PD model group rats are decreased and there may be different degrees of psychological anxiety.Subsequently,IHC was used to detect TH staining in the brain tissue of rats in the PD model group,and the results indicated that the number of TH staining positive cells in the striatum of rats in the PD model group decreased,indicating that the PD model was successfully constructed.In addition,the results of IHC also suggested that the number of TUNEL-stained positive cells and ROS levels in PD rats were increased,as well as the expressions of pyroptosis marker GSDMD,microglia activation marker IBA1,and the inflammation-related gene TLR4.WB results indicated that apoptosis molecular markers,cleaved caspase-3 and cleaved PARP,autophagy molecular markers LC3B and Beclin1,oxidative stress related gene NOX4,and protein expression of inflammatory factor TNF-αwere increased in the model family compared with the sham operation group.These results indicated that apoptosis,pyroptosis,autophagy,oxidative stress and inflammatory phenotypes increased in PD rat models.The m RNA expressions of NLRP3,caspase-1 and IL-1βwere increased in PD rat model by RT-PCR.IF the results showed that the protein expression of the above molecules increased in PD rat model;WB results showed that compared with sham operation group,NLRP3 inflammatory bodies,cleaved-caspase1,GSDMD-N and mature IL-1βwere increased in the striatum of PD rat model,suggesting an increase in pyroptosis in PD rat model.(2)Pyroptosis increased in PD cell models:Firstly,CCK-8 was used to detect the changes of PC12 cell viability under different concentrations of MPP~+and different treatment times to select the optimal modeling conditions.The results showed that with the increase of MPP~+concentration or treatment time,the viability of PC12 cells decreased.After 48h of MPP~+500μM treatment,the cell vitality of PC12 cells decreased to about 78%,while after 72h of treatment,the cell vitality was too low,decreasing to 52%.Therefore,we used MPP~+500μM treatment for 48h to build a PD cell model,and detected the TH protein expression in the model.Both immunofluorescence and WB results indicated that TH protein expression in PD cell model decreased,indicating that PD cell model was successfully constructed.Flow cytometry showed that compared with normal cells,the apoptotic cells in PD cell model increased.LDH release experiment showed increased LDH release in PD cell model.RT-PCR showed that NLRP3,caspase-1 and IL-1βm RNA expressions were increased in PD cell models.WB results showed that NLRP3,cleaved-caspase1,GSDMD-N and mature IL-1βwere increased in PD cell model.ELISA results showed that IL-1βprotein expression was increased in the supernatant of PD cell model.In conclusion,compared with normal cells,PD cell model showed an increased pyroptosis level.2.Up-regulated expression of IRF7 in PD model:Through RNA sequencing analysis of PD rat brain tissues,we found that the expression of innate immune-related genes,such as IRF7,ISG15,OAS1a,CXCL10,was up-regulated in PD model,and IRF7 was the hub protein most closely related to other genes in the protein interaction network.RT-PCR results showed that m RNA expression of IRF7increased in the striatum of PD rats.WB results showed that the protein expression of p-IRF7 in the striatum of the PD rat model was increased,confirming that the expression of IRF7 was up-regulated in the PD rat model.Subsequently,RT-PCR was used to detect the m RNA expression of IRF7 in PD cell models,and the results showed that the m RNA expression of IRF7 firstly increased and then decreased with the extension of MPP~+treatment time.Compared with the control group,m RNA expression of IRF7 was increased in PC12 cells treated with MPP~+500μM and MPP~+1000μM for 12h.The results of WB indicated that the protein expression of p-IRF7was significantly increased in PC12 cells treated with MPP~+500μM for 48h.3.IRF7 regulates pyroptosis in nerve cells through NLRP3:In the IRF7overexpression cell model,we observed decreased cell viability,increased LDH release,increased apoptosis rate,increased expression of pyroptosis molecular markers such as caspase1,GSDMD,IL-1β,and decreased TH protein expression.The m RNA and protein expressions of NLRP3 were also increased with the increase of IRF7.On the contrary,the expressions of NLRP3 and pyrogenic genes were significantly down-regulated with the knockdown of IRF7 in PD cell model,and the expression of TH protein was increased with the knockdown of IRF7.The results of double luciferase assay showed that the NLRP3 promoter activity was increased in IRF7 overexpression group compared with negative control group.4.TLR4/TAK1/IRF7 promotes neuronal pyroptosis through NLRP3:Further analysis of PD rat brain tissue chip data revealed that differentially expressed genes were significantly enriched in TLR signaling pathway,MAPK signaling pathway and other inflammatory signaling pathways.The results of WB detection in PD rat brain tissue and PD cell model indicated the activation of TLR4/TAK1 signaling pathway.After the TAK1 signaling pathway inhibitor(5Z)-7-Oxozeaenol was administered in PD cell models,it was observed that the TAK1 and IRF7 expressions were down-regulated and the NLRP3 expression was decreased significantly in PD cell models treated with 5μM concentration of(5Z)-7-Oxozeaenol for 1 hour.At the same time,increased cell viability,decreased apoptosis rate,decreased LDH release,decreased the expression of caspase1,GSDMD,IL-1βand other pyroptosis markers,and increased TH expression.Finally,the TAK1 signaling pathway inhibitor(5Z)-7-Oxozeaenol was added into the overexpressed IRF7 cell model,and the changes of IRF7 and NLRP3 expression and pyroptosis levels were observed.The results showed that overexpression of IRF7 partially reversed the effect of blocking TAK1 signaling pathway on NLRP3 expression and neurons pyroptosis.Conclusions:1.NLRP3 inflammatorome is significantly activated in PD cells and animal models,and can promote PD pathogenesis by inducing neuronal pyroptosis.Pyroptosis may be one of the way of dopaminergic nerve cell death.2.IRF7,as a key molecule of innate immune response,is up-regulated in PD cells and animal models,and may regulate its expression by binding to the NLRP3promoter,which is the molecular mechanism of nerve cell pyroptosis.3.TLR4/TAK1 signaling pathway,as the initial link of innate immune response,is significantly activated in PD cells and animal models,and can affect nerve cells’pyroptosis by regulating the expression of downstream IRF7 and NLRP3.The TLR4/TAK1/IRF7/NLRP3 axis may be a molecular target for PD therapy.