Effects Of Hydrogen Sulfide On Oxidative Stress Of Intestinal Epithelial Cells And Sulfide Oxidation Function Of Mitochondria

Hydrogen sulfide(H2S)is one of the most common intestinal microbial metabolites.When the concentration of H2S is too high,it will have an adverse effect on the body.However,the effect of high concentration of H2S on intestinal epithelial cell and its mitochondrial function remains unclear.This study first explored the effects of different concentrations of H2S on intestinal epithelial cells and mitochondrial functions,in order to determine the optimal H2S concentration for subsequent experiments.Then,based on the previous research,the effects of H2S on intestinal epithelial cell and its mitochondrial H2S detoxification,oxidative stress and so on under normal and inflammatory conditions were studied by means of transcriptome sequencing technology.Finally,through the silencing of H2S detoxification gene,the feedback regulations of mitochondrial dysfunction on H2S metabolism and oxidative stress of intestinal epithelial cells were explored.Therefore,the relationship between H2S and intestinal epithelial mitochondria is comprehensively summarized,which provides theoretical support for targeting microbiota and mitochondria to regulate intestinal health.1.Effects of different concentrations of hydrogen sulfide on intestinal epithelial cells and its mitochondrial functionIn this study,Caco-2 cells and sodium hydrosulfide(NaHS,H2S donor)were used to study the effects of H2S on intestinal epithelial cell inflammation,oxidative stress and mitochondrial sulfide detoxification,and to explore the possible mechanism of H2S affecting intestinal health.The test was divided into four groups,which were the control group and the low(100 μM NaHS),medium(1 mM NaHS)and high(2 mM NaHS)concentrations of NaHS group.The treatment time of the four groups was 24 h.The results showed that NaHS enhanced cell viability.2 mM NaHS significantly up-regulated the gene expression levels of tumor necrosis factor alpha(TNF-α).100 μM NaHS up-regulated the gene expressions of the mitochondrial sulfide detoxification enzymes sulfide quinone oxidoreductase(SQR),thiosulfate sulfur transferase(TST),sulfur dioxygenase(ETHE1)and sulfite oxidase(SUOX).However,2 mM NaHS down-regulated the gene expression of SQR.2 mM NaHS significantly increased malondialdehyde(MDA),glutathione(GSH)levels,glutathione peroxidase(GPx)activity and significantly reduced superoxide dismutase(SOD)activity.100 μM NaHS significantly increased MDA level.1 mM NaHS significantly reduced SOD activity.Low concentration of H2S enhances cell viability,the mitochondrial sulfides detoxification ability and oxidative stress.High concentration of H2S promotes inflammatory response,provokes oxidative stress,affects the mitochondrial sulfides detoxification ability,and harms intestinal health.2.Regulation of hydrogen sulfide on intestinal epithelial cells and mitochondria in normal and inflammatory statesThis experiment aimed to explore the effects of H2S on intestinal epithelial cell inflammation,oxidative stress,H2S detoxification and mitochondrial function under normal and inflammatory conditions by means of transcriptome sequencing technology.The test was divided into four groups:the control group did not do any treatment,the NaHS group was treated with 2mM NaHS for 24 h,the LPS group was treated with 1 μg/mL LPS for 24 h,and the LPS+NaHS group was first treated with 1 μg/mL LPS for 24 h and then treated with 2mM NaHS for 24 h.They constituted a comparison of normal and inflammatory conditions of intestinal epithelial cells respectively.There are 3-6 replicates in each group.The results are shown below:NaHS significantly enhanced the viability of intestinal epithelial cells.NaHS significantly upregulated the gene expression of TNF-α in normal intestinal epithelial cells.NaHS significantly increased GSH level and GPx activity in cells.NaHS significantly inhibited the gene expression of H2S detoxification enzyme SQR.NaHS significantly upregulated the protein expression level of autophagy related microtubule-associated proteinl lightchain3(LC3II)and the gene and protein expression levels of Bcl-2/adenovirus E1B 19-kDa-interacting protein 3(BNIP3).NaHS significantly reduced mitochondrial DNA(mtDNA)gene expression and caused mitochondrial swelling.In conclusion,high concentration of H2S can enhance the cell viability of intestinal epithelial cells,promote the occurrence of inflammation,increase the oxidative stress level of cells,inhibit the activity of key enzyme in H2S detoxification pathway of mitochondria and reduce the detoxification ability of H2S,strengthened autophagy and mitophagy,and affect the morphology and function of mitochondria.3.Effects of mitochondrial dysfunction on hydrogen sulfide metabolismIn this experiment,gene silencing was used to investigate the feedback regulation of mitochondrial dysfunction on H2S metabolism and oxidative stress in intestinal epithelial cells.In this experiment,the key enzymes SQR and ETHE1 in H2S detoxification process were inhibited respectively,and their effects on Caco-2 cell inflammation,oxidative stress and mitochondrial H2S detoxification were studied.The test was divided into five groups:the control group did not do any treatment,the lipo group only added lipo transfection reagent,the lipo+NaHS group was first treated with lipo and then treated with 2mM NaHS for 24 h,the NaHS group was treated with 2mM NaHS for 24h,and the interference(lipo+siRNA+NaHS)group was first transfected siRNA into cells with lipo and treated with 2mM NaHS for 24 h.There are 5 replicates in each group.The results are shown below:Inhibitions of SQR and ETHE1 attenuated cell viability compared to the NaHS group.Compared with the NaHS group,inhibition of SQR increased GSH level and enhanced the activity of GPx in cells,inhibition of ETHE1 reduced GSH level and GPx activity in cells.Inhibition of SQR and ETHE1 had no significant effect on TNF-α gene expression compared with the NaHS group.Compared with NaHS group,inhibition of SQR downregulated SUOX gene expression,inhibition of ETHE1 significantly upregulated SUOX gene expression.Inhibitions of SQR and ETHE1 had no significant effect on BNIP3 gene expression compared to the NaHS group.The above results indicate that mitochondrial dysfunctions caused by H2S detoxification gene silencing affect the downstream H2S detoxification process and cellular oxidative stress state,indicating that mitochondrial function stability is critical for H2S detoxification process and cell state.In summary,high concentration of H2S will enhance the cell viability of intestinal epithelial cells,promote the occurrence of inflammation,increase the oxidative stress level of cells,reduce the detoxification ability of H2S,strengthen mitophagy and affect the morphology and function of mitochondria.The mitochondrial function is also important for cell status and H2S detoxification.