Anti-inflammation Effect And The Mechanism Of Suitable Concentration Of Short-chain Fatty Acids Mixture On Microglia

Neuroinflammation is the main cause of many central nervous system(CNS)diseases,which microglia(unique immune cells of CNS)play the most important role in neuroinflammation.Pathogenic microbe,physical trauma and abnormal aggregation of proteins,etc.all can activate microglia causing a strong neuroinflammatory reaction,which in turn causes damage to neurons and ultimately lead death of neurons.The diseases of CNS like Parkinson’s disease(PD),Autism spectrum disorder,Alzheimer’s disease are closely related to neuroinflammation.In recent years,the relationship between the gut microbiota and CNS has been widely concerned.Gut microbiota can act on the CNS through the gut-brain axis which mainly contains four pathways of gut microbiota metabolite,endocrine,vagus nerve and immunity.The disorders of gut microbiota involve a variety of CNS diseases,for example,studies on gut microbiota in PD had found that PD patients not only had disorder of gut microbiota,but also had significant changes in short-chain fatty acids(SCFAs)which are the main metabolites of gut microbiota;animal experiments had found that microglia in the brain of germfree mice have abnormal morphology and function,but feeding SCFAs to germfree mice can correct the abnormalities of microglia.The above suggests the correlation between SCFAs and neuroinflammation.Based on these,we explored the effects of SCFAs on microglia in inflammatory environment,and further explored the role of SCFAs in neuroinflammatory response and its potential mechanism.We used lipopolysaccharide(LPS)to stimulate mouse microglia(BV-2 cells)to obtain a model of neuroinflammation.BV-2 cells were randomly divided into a control group(Control Group),model group(LPS),drug-administered group(LPS+SCFAs mix),and drug control group(SCFAs mix)to study the effects of SCFAs mixture on microglia.First we used the CCK-8 kit to detect the viability of BV-2 cells treated with different concentrations of sodium acetate,sodium propionate,and sodium butyrate to determine the appropriate "working concentration" of three SCFAs which have no effect on cell viability,and three SCFAs constituted the SCFAs mixture(SCFAs mix)at their respective working concentrations which had also does not affect on cell viability.Nitric oxide(NO)concentration in cell supernatants was detected by NO kit.The results showed that SCFAs mix with unsuitable concentration(mimicking the concentration of SCFAs in fecal bacteria of PD model mice)significantly increased the production of NO in LPS-stimulated BV-2 cells and suitable concentration of SCFAs mix significantly inhibited the production of NO in LPS-stimulated BV-2 cells,however,a single SCFA at the same working concentration could not inhibit the production of NO in LPS-stimulated BV-2 cells.Results of enzyme-linked immunosorbent assay(ELISA)showed that suitable concentration of SCFAs mix can inhibit the production of NO,TNF-α and IL-6 in LPS-stimulated BV-2 cells.Quantitative real-time PCR(qPCR)experiments showed that SCFAs mix inhibited the inflammatory response of LPS-stimulated BV-2 cells by reducing the expression of TNF-α,IL-6,iNOS and NLRP3 mRNA in BV-2 cells.In order to detect the potential mechanism,we examined the expression of inflammatory pathway factors such as TLR4,MyD88,TRAF6 and NF-κB at the protein level by Western blot.The results indicate that the suitable concentration of SCFAs mix inhibits inflammatory response of microglia by inhibiting TLR4/MyD88/TRAF6/NF-κB inflammatory pathway.SCFAs need to rely on their transporter and receptors to exert biological activity.To further explore changes in SCFAs associated with neuroinflammation-related CNS diseases,changes in their transporter and receptors,we established subacute PD mouse model by intraperitoneal injection 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine(MPTP)to mouse.The number of dopaminergic neurons in substantia nigra of mice were detected by immunofluorescence(IF).Results of IF showed that number of dopaminergic neurons in substantia nigra of MPTP-induced PD mice was significantly reduced than control group.The level of dopamine in striatum of mice was detected by high performance liquid chromatography(HPLC).Results of HPLC showed that the level of dopamine in striatum of MPTP-induced PD mice was significantly reduced than control group.The expression of SCFAs transporter Slc5a8,receptors GPR43 and GPR109 a in mouse colon and cerebral cortex were detected by qPCR.Results of qPCR showed that the expression of Slc5a8 in colon of MPTP-induced PD mice was significantly higher than control group,while the expression of GPR43 and GPR109 a did not change compared with the control group;however,the expression of GPR43 receptor was significantly decreased,the expression of Slc5a8 and GPR109 a was not significantly changed in cerebral cortex of PD model mice.This indicates that SCFAs may regulate PD by regulating the expression of transporter and receptors of SCFAs.