Effect Of Mitochondria-targeted Antioxidant SS-31 On Cigarette Smoke-induced Airway Inflammation And Oxidative Stress

Objective: Excessive production of reactive oxygen species induced by cigarette smoke triggers and promotes chronic airway inflammation and protease/antiprotease imbalance,which plays an important role in airway inflammatory diseases such as chronic obstructive pulmonary disease(COPD).Systematic studies on cigaretteinduced airway inflammation and oxidative stress are expected to find new targets and new drug interventions for cigarette-induced airway inflammatory diseases such as COPD.As a mitochondrial targeted antagonist,Szeto-schiller peptide-31(SS-31)has potential clinical application value on pulmonary inflammatory diseases due to its potent capacity of anti-oxidant and anti-inflammation and relative safety.However,few studies about the application of SS-31 to pulmonary diseases have have been reported and little is known regarding whether this drug is effective for cigarette-related disease intervention.In the present study,using cells and mice experimental models,we amid to explored whether mitochondria-targeted peptide SS-31 plays a protective role in cigarette smoke(CS)-induced airway inflammation and oxidative stress and its underlying mechanisms in vivo and in vitro.Materials and Methods: Chapter 1: Effects of SS-31 on cigarette induced airway inflammation and pulmonary oxidative stress in mice Established airway inflammation and oxidative stress model by fumigating mice: Mice were divided into four groups(n = 10 per group): control group(CON);cigarette smoke-exposed group(CS);CS-exposed and low-dose SS-31 group(2.5mg/kg)[CS + SS-31(L)],CS-exposed and high-dose SS-31 group(5mg/kg)[CS+ SS-31(H)].Mice were pretreated by SS-31 once via intraperitoneal injection,After 4 weeks exposure to CS,samples were collected.Bronchoalveolar lavage fluid(BALF)was applied to examine inflammatory cell counts.Inflammatory cytokines in BALF(interleukin 6/ tumor necrosis factor α/ matrix metallopeptidase 9)were examined by enzyme-linked immunosorbent assay(ELISA).Lung tissue homogenate was used to examine oxidative stress(malondialdehyde / superoxide dismutase / myeloperoxidase)via biochemical kit.Hematoxylin and eosin solution(H&E)、Alcian blue-periodic acid Schiff staining(AB-PAS)、 immunohistochemistry staining were used to assess airway pathological changes、airway mucus secretion status and the expression of MUC5 AC,respectively.Chapter 2: Mechanisms of SS-31 on cigarette induced airway inflammation and oxidative stress In order to further explore the mechanism of SS-31,RNA sequencing from lung tissue of mice was used to detect differentially expressed genes(DEGs).Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were applied to investigate the pathway which SS-31 be likely to participate in.Western blotting was used to investigate the expression changes of mitochondrial fusion protein-optic dystrophy protein 1(OPA1)and mitochondrial fission proteinmitochondrial fission factor(MFF)in mouse lung tissues,as well as the expression changes of related pathway proteins in each group.Chapter 3: Studies on the anti-inflammatory and anti-oxidative effects of SS-31 on MAPK signaling pathway Airway epithelial cell,BEAS-2B were cultured in vitro and then divided into five groups: control group(CON),cigarette smoke extract exposed-group(CSE),CSE+ani(anisomycin)group,CSE+ SS-31(100μM)group and CSE+SS-31+ani.Cells were pretreated with SS-31 for 1h,anisomysin was given preferentially to SS-31 for 1h,then exposed to CSE.After 24-hour incubation,inflammatory cytokines were examined in supernate by ELISA.Cell pellets was used to detect the levels of oxidative stress(MDA/SOD)by biochemical kit.Western blotting was used to further verify the inhibitory effect of SS-31 on MAPK pathway and detect the changes of OPA1 and MFF expression in cell pellets.Statistical analysis:All values are expressed as means ± the standard deviation.One-way analysis of variance was applied for statistical analysis,then the least significant difference test was used for multiple comparisons.Data were analyzed and figures were prepared using Graph Pad Prism 7(Graph Pad Software,San Diego,CA,USA).p < 0.05 was deemed to indicate statistical significance,p < 0.01 was considered as significant statistical differences.Results: Chapter 1: Effects of SS-31 on cigarette induced airway inflammation and pulmonary oxidative stress in mice 1)Hematoxylin and eosin solution staining showed SS-31 significantly attenuated cigarette smoke-induced mouse lung histologic changes and airway inflammation scores(p < 0.0001,equally),and the intervention of the high-dose group was significantly better than that of the low-dose group(p < 0.01);2)Alcian blue-periodic acid Schiff staining demonstrated high-dose SS-31 suppressed CS-induced airway mucus hypersecretion(p < 0.05);3)Immunohistochemistry staining showed high-dose SS-31 significantly suppressed CS-induced airway mucin 5AC secretion(p <0.01); 4)Inflammatory cell counts in BALF: SS-31 decreased the number of total cell number(low dose group: p < 0.01;high dose group:p<0.05)、neutrophils(low dose group:p<0.05)and macrophages(p < 0.0001,equally);5)The levels of inflammatory cytokines in BALF: high-dose SS-31 significantly reduced the levels of IL-6,TNF-α and MMP-9 in BALF induced by CS(p<0.0001,p < 0.01,p <0.0001 respectively);the levels of IL-6 and MMP-9 in high dose group is significantly lower than in low dose group(p < 0.01,< 0.001 respectively);6)Oxidative stress in lung homogenate:high-dose SS-31 significantly reduced the levels of MDA,MPO and increases the activity of SOD(p<0.01,p<0.001,p<0.0001,respectively).The effect of high-dose SS-31 on MPO and SOD was better than that of low-dose group(p < 0.05,< 0.01 respectively).Chapter 2: Mechanisms of SS-31 on cigarette induced airway inflammation and oxidative stress 1)Results of RNA sequencing and bioinformatic analysis RNA sequencing revealed there were 4038 differentially expressed genes(DEGs)induced by cigarette smoking,Upregulated 2034 DEGs,downregulated 2004 DEGs,respectively.On gene ontology analysis,those DEGs were mainly enriched in the biological processes of negative regulation of apoptotic process,cilium assembly,epithelial cilium movement,extracellular matrix organization.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis primarily showed enrichment in mitogen-activated protein kinase(MAPK)、Cyclic adenosine monophosphate(c AMP)、 Adenosine5‘-monophosphate-activated protein kinase(AMPK)signaling pathways,and extracellular matrix-receptor interactions.2)SS-31 improved mitochondrial dysfunction induced by CS in mice lung tissue High-dose SS-31 still upregulated expression of mitotic protein MFF(p<0.05)and downregulated the expression of mitochondrial fusion protein OPA1(p<0.05)induced by CS.3)SS-31 inhibited the activation of MAPK signaling pathway induced by CS in mice lung tissue High-dose SS-31 still inhibited the phosphorylation of ERK and P38 activated by CS(p< 0.01,p< 0.05 respectively).Chapter 3: Study on the anti-inflammatory and anti-oxidative effects of SS-31 on MAPK signaling pathway 1)SS-31 inhibited the activation of MAPK signaling pathway induced by CSE in BEAS-2B cells.Anisomycin further enhanced the phosphorylation of P38 in BEAS-2B cells induced by CSE(p<0.05),and SS-31 respectively inhibited the phosphorylation of p-38 induced by CSE or CSE+ani(p<0.05,p<0.01).2)SS-31 reduced the release of intracellular inflammatory cytokines induced by CSE via inactivation MAPK signaling pathway.Anisomycin further enhanced the release of IL-6,TNF-α and MMP-9 in BEAS-2B cells induced by CSE(p<0.001,p<0.0001,p<0.05),and SS-31 respectively inhibited the release of inflammatory cytokines in BEA-2B cells induced by CSE or CSE+ ani,The levels of IL-6,TNF-α and MMP-9 in CSE+SS-31 group were lower than those in SS-31+CSE+ ani group(p<0.05,p<0.05,p<0.01).3)SS-31 relieved the imbalance of intracellular oxidative stress induced by CSE via inactivation of MAPK signaling pathway(1)MDA: Anisomycin further increased the MDA production in CSE-induced BEAS-2B cells(P <0.01),SS-31 respectively inhibited the MDA production in CSE group or CSE+ ani group(p<0.05,p<0.01),the content of MDA in SS-31+CSE group was significantly lower than that in SS-31+CSE + ani group(P <0.01).(2)SOD: The activity of SOD significantly decreased induced by CSE or CSE+ani in BEAS-2B cells(p<0.0001,p<0.0001),SS-31 respectively improved the activity of SOD in CSE group or CSE+ ani group(p<0.05,p<0.01.4)SS-31 improved CSE-induced mitochondrial dysfunction in BEAS-2B cells(1)OPA1: CSE or CSE+ ani down-regulated the expression of OPA1 in BEAS-2B cells respectively(P <0.001,P <0.0001),SS-31 reversed those changes above.(p<0.05,p<0.05).(2)MFF: CSE or CSE+ ani up-regulated expression of MFF in BEAS-2B cells,respectively(p<0.01,p<0.001).SS-31 reversed those changes above(p<0.05,p<0.01).Conclusion: SS-31 alleviates CS-induced airway inflammation and oxidative stress,possibly via the regulation of mitochondrial function and MAPK pathway,SS-31 may be a novel drug for CS-induced airway disorders.