Effect And Mechanism Of Endogenous Hydrogen Sulfide On Lipopolysaccharide-induced Inflammation Of Rat Small Intestinal Enteric Glial Cells

Aim:The pathogenesis of chronic intestinal inflammatory diseases including Inflammatory bowel diseases（IBD）and Irritable bowel syndrome（IBS）is related to intestinal mucosal barrier dysfunction,inflammation,intestinal flora disturbance,visceral hypersensitivity,etc.Among them,immune cells release inflammatory mediators and damage the function of the Enteric nervous system（ENS）,which can lead to a series of neuropathological and physiological changes,including increased visceral sensitivity changes.Enteric glial cells（EGCs）,an important part of the ENS,are suitable cell lines for studying intestinal neurogenic inflammation.Hydrogen sulfide（H₂S）,a kind of gaseous signaling molecule,is involved in nerve cell signaling transduction and various functional adjustments of the digestive system,but its mechanism for regulating intestinal neurogenic inflammation is still unclear.Lipopolysaccharides（LPS）was used to induce the inflammation of EGCs in rats,simulating intestinal neurogenic inflammatory response.Based on this,this study also explores the effect and mechanism of endogenous H₂S on EGCs under the inflammatory damage,which provides theoretical support for further elucidating the relationship between chronic intestinal inflammatory diseases and EGCs,as well as the regulatory mechanism of endogenous H₂S.Methods:1.The expressions of H₂S-producing enzymes Cystathionine-γ-lyase（CSE）and Cystathionine-β-synthase（CBS）in EGCs were detected by immunofluorescence staining.2.In order to screen the concentration and duration of LPS,EGCs were stimulated with different concentrations of LPS for 6 h,24 h or 48 h.Then the cell viability was detected by Cell Counting Kit-8（CCK-8）,the state of cells was observed by light microscope,and the m RNA level of IL-6 was measured by Real-time quantitative polymerase chain reaction（RT-q PCR）.3.EGCs were treated with different concentrations of L-cysteine（L-Cys,CSE and CBS substrate）,D,L-propargylglycine（PAG,CSE inhibitor）or Aminooxyacetic Acid（AOAA,CBS inhibitor）for 25 hours.The cell viability was detected by Methylthiazolyldiphenyl-tetrazolium bromide（MTT）and the cell state was observed by light microscope to determine the action time and screen the concentrations of L-Cys,PAG or AOAA.4.EGCs under the inflammatory damage induced by LPS were treated with L-Cys,PAG or AOAA.EGCs were divided into Control group,LPS group,LPS+L-Cys-L/LPS+PAG-L/LPS+AOAA-L group and LPS+L-Cys-M/LPS+PAG-M/LPS+AOAA-M group.7-Azido-4-methylcoumarin（Az Mc）fluorogenic probe was used to detect the H₂S content.CSE and CBS m RNA and protein expression levels were detected by RT–q PCR and Western Blotting.MTT was used to detect cell activity.Light microscope was used to observe cell state.RT-q PCR and Enzyme-linked immunosorbent assay（ELISA）were used to detect IL-6,IL-1β,TNF-αand i NOS m RNA and protein expression.And Western Blotting（WB）was used to detect the NF-κB pathway p65,p-p65,IκB,p-IκB protein expressions.Results:1.Immunofluorescence staining showed that EGCs expressed CSE and CBS proteins.2.CCK-8/MTT results showed that LPS,L-Cys,PAG or AOAA concentration-dependently decreased the cell viability and the number of EGCs.After LPS induced inflammation,the use of L-Cys can restore the activity of EGCs and increase the number of EGCs,while PAG or AOAA could reduce the activity and number of EGCs.3.Az Mc fluorescence probe results showed that LPS could reduce the H₂S content in EGCs compared to Control group;Compared with LPS group,L-Cys increased intracellular H₂S content,while PAG or AOAA decreased intracellular H₂S content.4.RT-q PCR results showed that compared with Control group,the m RNA expressions of CSE and CBS were down-regulated,while the m RNA expressions of IL-6,IL-1β,i NOS and TNF-αwere up-regulated in LPS group;Compared with LPS group,L-Cys up-regulated the m RNA levels of CSE and CBS in EGCs and down-regulated the m RNA levels of IL-6,IL-1β,i NOS and TNF-α;PAG or AOAA decreased the m RNA levels of CSE or CBS in EGC_S and increased the m RNA levels of IL-6,IL-1β,i NOS and TNF-α.5.ELISA results showed that compared with Control group,the expressions of IL-6,IL-1βand TNF-αin culture supernatant were up-regulated;Compared with LPS group,the expressions of IL-6,IL-1βand TNF-αin culture supernatant were decreased in LPS+L-Cys goups which were increased in LPS+PAG groups and LPS+AOAA groups.6.WB results showed that compared with Control group,the protein expressions of CSE and CBS in LPS group were down-regulated,and p-p65/p65 and p-IκB/IκB of NF-κB pathway were elevated;Compared with LPS group,the protein levels of CSE and CBS in EGCs were increased and p-p65/p65 and p-IκB/IκB were decreased in LPS+L-Cys groups;In LPS+PAG groups,CSE protein expression was down-regulated,CBS protein expression was up-regulated,p-p65/p65 and p-IκB/IκB were elevated;In LPS+AOAA groups,CBS protein expression was down-regulated,CSE protein expression was up-regulated,p-p65/p65 and p-IκB/IκB were elevated.Conclusions:1.The H₂S-producing enzymes CSE and CBS are expressed in rat small intestine EGCs.2.LPS-induced inflammation can lead to the decrease in the number and viability of EGCs,the down-regulation of CSE and CBS m RNA and protein expressions,the reduction of intracellular H₂S content,and the up-regulation of pro-inflammatory factors IL-6,IL-1β,i NOS and TNF-αm RNA and protein expressions.3.L-Cys can increase the m RNA and protein levels of CSE and CBS in EGC_S,increase the number and activity of EGC_S,and decrease the pro-inflammatory factors IL-6,i NOS,IL-1βand TNF-αexpression levels by up-regulating endogenous H₂S;PAG can down-regulate the expressions of CSE and endogenous H₂S,increase the m RNA and protein levels of CBS,decrease the number and activity of EGC_S,and increase the expression levels of pro-inflammatory factors IL-6,i NOS,IL-1βand TNF-α;AOAA can down-regulate the expressions of CBS and endogenous H₂S,increase the m RNA and protein levels of CSE,decrease the number and activity of EGC_S,and increase the pro-inflammatory factors IL-6,i NOS,IL-1βand TNF-αexpressions.4.Endogenous H₂S may play a role in LPS-induced EGC_S inflammation in rat small intestine through NF-κB signaling pathway.LPS can increase p65 and IκB phosphorylated proteins,namely p-p65 and p-IκB,expressions,L-Cys can decrease p-p65 and p-IκB expressions by upregulating endogenous H₂S.Both downregulating of CSE by PAG and downregulating of CBS by AOAA can down-regulate endogenous H₂S,which can increase p-p65 and p-IκB protein expressions.