| Sulfur mustard (SM) is a typical blister agents. Exposure to SM results inincapacitating health effects of multiple organs and systems followed by remarkablelong-term complications. Skin tissue is the primary target of SM exposure, where itinduces early poisoning damage including erythema, edema, vesication and purpuras.The most common symptoms of chronic injury are dry skin with persistent pruritis,burning, desquamation and cherry angioma. Due to complex toxicological mechanisms,there is no cure available against SM injury.SM-induced tissue injury and inflammation are closely related. SM inducedneutrophils, monocytes and macrophages infiltrating into injured tissue. These cellsrelease myeloperoxidase (MPO) and cytokine IL-1β, IL-6, TNF-α, MCP-1, IL-8andthen induce the increase of substance P, plasminogen activator, histamine. Manyinflammation-related pathways involved in SM injury, including arachidonic acidmetabolic pathway, mitogen-activated protein kinase (MAPK) signaling pathways,nuclear transcription factor-κB (NF-κB) pathways. Arachidonic acid producesprostaglandins and leukotrienes by cyclooxygenase and lipoxygenase catalysing.MAPK pathway and NF-κB pathway could affect the transcription of inflammatorymediators. The targets research based on the key molecules in the signaling pathwaymentioned above plays a critical role in drugs research against SM injury.Depending on different regulation mechanism of SM-induced inflammation, Ourstudy chose seven kinds of anti-inflammatory and immunosuppressant to evaluate theprotective effects on SM-induced cutaneous injury, including steroidal anti-inflammatorydrug, non-steroidal anti-inflammatory drug (NSAID), capsaicin receptor agonist, p38MAPK inhibitors, necroptosis inhibitors, immunosuppressant and so on. This researchmight contribute to understanding the characteristics of different types ofanti-inflammatory drugs and providing a reliable experimental basis forinflammation-based mechanism and drug research.. Objective:Our aim is to evaluate the protective effects of different anti-inflammtory drugs andcombination regimen against SM-induced injury.Methods:Establishment of Mouse Ear Vesicant Model (MEVM): male KM mice right earswere exposed to5μL of32or128mg/mL SM, respectively. After24or48h SMexposure, ear tissue was harvested and weighted and histopathological analysis wasconducted;Establishments of SM systemic poisoning model: male BalB/c mice were injectedsubcutaneously by SM (40mg/kg), survival of animals exposed to7days post exposurewere observed;Establishments of SM injury model in vitro: human epidermal keratinocytes(HaCaT cells) were exposed with200,1000μM SM for30min, then washed by PBSsolution and further cultivated in the presence of each compound for24h. Cell viabilityand IL-6and IL-8in cell supernatant were detected;Screening model for p38inhibitor compounds in vitro: murine macrophage cell lineRAW264.7cells were stimulated by1μg/mL LPS in the presence of each compoundfor24h, AlphaLISA was used to detect TNF-α concentration;Time course of SM-induced activation of p38and JNK: HaCaT cells were exposedwith200,1000μM SM for30min, then were washed by PBS solution and furthercultivated for0,0.5,1,1.5,3,6,12,24h. Luninex, multifunctional liquid chip technologyanalyzes system, was used to detect the levels of p-p38, t-p38, p-JNK and t-JNK incell lysis.Effects of SB203580and BIRB796on SM-induced p38and JNK activation:HaCaT cells were exposed with200,1000μM SM for30min, then washed by PBSsolution and further cultivated in the presence of20μM SB203580and BIRB796for0.5and12h. Luninex technology and Western Blotting were used to detect the levels ofp-p38, t-p38, p-JNK and t-JNK in cell lysis..Results:1. The protective effects of steroidal and non-steroidal anti-inflammatory drug againstSM injury1.1. Establishment of MEVM induced by different concentration of SM The degree of skin damage depends on concentration of SM, the higher exposuredose, the higher degree of swelling of mouse ear skin. The commonly used exposuredoses,32mg/mL SM5μL (low dose), and the maximum solubility of SM in solvent,128mg/mL SM5μL (high dose) were used in this model to establish Mouse EarVesicant Model of different damage degrees.1.2. The effect of steroidal anti-inflammatory drug dexamethasone against SM-inducedinjuryOrally administration of dexamethasone (5mg/kg) to mice30min prior to SMexposure had no effect on SM-exposed mouse ear edema.1.3. The effect of NSAID indomethacin against SM-induced injuryOrally administration indomethacin (10mg/kg) to mice30min prior to SMexposure could significantly inhibit the high and low doses of SM-induced mice earedema by40%and25.68%, respectively.2. The effects of o new drugs with anti-inflammatory activity against SM injury2.1. The effect of the capsaicin receptor agonist olvanil against SM-induced injuryIn our study, olvanil (0.125mg/ear,0.25mg/ear and0.05mg/ear) were topicallyadministrated in the inner right mice ear, MEVM exposed by different concentration ofSM was used to evaluate the effects of olvanil. NSAID indomethacin (10mg/kg) wasused as a positive control. The results show that the olvanil significantly inhibitedmouse ear edema exposed to low doses of SM, and showed stronger inhibitory effectthan indomethacin, but had no effect on ear edema induced by the high dose of SM.H&E staining were used to analysis the effect of olvanil on histopathology of SMinduced skin injury. The results show that, in olvanil group, necrosis and inflammatorycell infiltration were restored. Olvanil showed stronger protective effects thanindomathacin.2.2. The effect of p38inhibitors against SM-induced injury2.2.1. Screening of BIRB796analoguesLPS-stimulated RAW264.7cells model was used to screen the inhibitive effect of50BIRB796analogues on TNF-α secretion. P38inhibitor SB203580was used as apositive control. The results showed that the compounds p38-98, p38-99, p38-123can significantly inhibit LPS-induced TNF-α secretion with low cytotoxicity, IC50was10.33μM,48.92μM and19.39μM.2.2.2. The effects of p38inhibitors against SM injury in vitro2.2.2.1. The effects of p38inhibitors on SM-induced IL-6and IL-8in HaCaT cellsIn SM-exposed HaCaT cells model, the inhibitive effects of the p38inhibitorSB203580, BIRB796and p38-98, p38-99on inflammatory cytokines IL-6andchemokines IL-8secretion were observed. The results showed that, SB203580,BIRB796, p38-98,99can significantly inhibit the secretion of IL-6and IL-8in a gooddose-dependent manner. Between two representatives of the p38inhibitor, BIRB796showed stronger inhibitory effect than SB203580.2.2.2.2. The effects of p38inhibitors on SM-exposed HaCaT cell survivalSM-exposed HaCaT cell model was used to observe protective effect of the p38inhibitor SB203580, BIRB796p38-98,p38-99. The results showed that, SB203580,BIRB796, p38-98,99had a protective effect on SM-exposed cells6h post-exposure.2.2.3. The effects of p38inhibitors against SM injury in vivo2.2.3.1. The effect of p38inhibitors against SM-induced skin injuryp38inhibitor SB203580, BIRB796and active compounds p38-98,99(10mg/kg)were intraperitoneally administrated to mice at24h before exposure,30min and12hafter exposure. The results showed that, apart from SB203580, BIRB796, p38-98,99showed an inhibitive trend on inhibition auricle edema.2.2.3.2. The effect of p38inhibitors against SM-induced systemic poisoning modelP38inhibitor SB203580, BIRB796, p38-98,99(5mg/kg/d) were intravenouslyadministrated to male BalB/c mice. SM (40mg/kg) was injected subcutaneously to mice,the survival condition of animals within7days post exposure was observed. The resultsshow that, in the model group, mice all died within five days post-exposure, the druggroup mice were exposed all died in the sixth or seventh days. P38inhibitors groupshowed no effects on SM-induced mice death.2.2.4. Metabolic stability study of p38inhibitor compoundsRat liver microsomes model was used to observe the metabolic stability ofSB203580, BIRB796, p38-98,99. The results showed that p38-98has good metabolic stability.2.2.5. Mechanism analysis of different p38inhibitors against SM injuryIn order to elucidate the involvement of inflammation-relevant SAPKs activityaffected by the two kinds of p38MAPK inhibitors upon exposure to SM, the activationof p38and JNK in HaCaT cells were examined2.2.5.1. Effects of SM on p38and JNK phosphorylation in HaCaT cellsLuminex assay was used to investigated a time course of JNK and p38activated by200μM SM. The results showed SM exposure provoked a rapid increase in both p38and JNK phosphorylation in30min after exposure2.2.5.2. Effects of p38inhibitors on SM-induced activity of p38and JNK in HaCaTcellsTwo methods: western blotting and Luminex assay,were used to further analyze theeffects of two kinds of p38MAPK inhibitors, SB203580and BIRB796, onphosphorylation of p38and JNK. Results showed that BIRB796simultaneouslyinhibited SM-induced activation of p38and JNK, while SB203580inhibited p38butenhance JNK activation.Collectingly, p38inhibitors have a significant protective effect on SM-exposedcell model and a potential protect effect on SM-induced skin injury. Compared withSB203580, BIRB796might be more efficient in alleviating SM-induced cutaneousinjury, which may because BIRB796simultaneously inhibited SM-induced activation ofp38and JNK, while SB203580inhibited p38but enhance JNK activation.2.3. The effect of necroptosis inhibitors Nec-1against SM-induced injury2.3.1. The effect of necroptosis inhibitors Nec-1against SM-exposed HaCaT cellsurvivalSM-exposed HaCaT cell model was used to observe the effects of Nec-1combinedwith apoptosis inhibitor z-VAD-fmk on cell survival. The results showed, Nec-1,z-VAD-fmk showed significantly protective effect. And the cell survival rate nearlyreached100%in the combined group. It indicated that necroptosis involved inSM-induced cells death.2.3.2. The effect of necroptosis inhibitors Nec-1against SM injury Intraperitoneal administration of Nec-1(4mg/kg) and z-VAD-fmk (10mg/kg) tomice before exposure had no significant effect on the SM-induced ear edema.2.4. The effect of immunosuppressant FTY720against SM-induced injuryContinuous orally administration of S1P inhibitors FTY720(1mg/kg/d) for3dayshad no effect on SM-exposed mice ear edema.2.5. The effect of aescinate sodium against SM-induced injuryIn this study, SM-induced skin injury model was used to investigate the effects oftraditional Chinese medicine preparation aescine gel and injection of sodium aescin.The results showed that topical application sodium aescine gel or injectionintraperitoneal injection of aescin (5mg/kg) had no effect on SM-induced ear edema.3. The protective effects of anti-inflammatory drugs in combination with other drugsagainst SM injuryMany mechanism involve in SM-induced injury, so simple drugs shows limitedeffect. Therefore, to improve the effectiveness of drug treatment, it needs to combinedifferent drugs based on different mechanisms to build comprehensive combinationmeasures. In this study, we investigate the protective effect of anti-inflammatory drugsin combination with PARP inhibitors against SM injury.3.1. Effects of indomethacin combined with PRAP inhibitor on SM-exposed MEVMUsing SM-exposed ear mouse model to evaluate the protective effects ofnon-steroidal anti-inflammatory drug indomethacin (10mg/kg, po) combined with PARPinhibitors MUS003(200mg/kg, ip). The results showed, in low doses of SM exposureconditions, indomethacin group and drug combination group could reduce the earswelling, no interaction between the two drugs. In high doses of SM exposureconditions, MUS003, indomethacin group, drug combination could reduce the earswelling, no interaction between the two drugs.3.2. Effects of olvanil combined with PRAP inhibitor on SM-exposed MEVMSM-exposed ear mouse model was used to evaluate the protective effects ofcapsaicin receptor agonist olvanil (0.5mg/ear, topically) combined with PARPinhibitors MUS003(200mg/kg, ip). The results showed, in low doses of SM exposureconditions, olvanil group and drug combination group could reduce the ear swelling,there is significant interaction between the two drugs. In high doses of SM exposure conditions, MUS003and drug combination could reduce the ear swelling, no interactionbetween the two drugs.Conclusion：1Non-steroidal anti-inflammatory drug indomethacin obviously inhibited thecutaneous injury induced by32mg/mL and128mg/mL SM. Capsaicin receptoragonist olvanil significantly prevented the low doses of SM-induced skin damage,and showed more effective effect than indomethacin;2Compared with the competitive p38inhibitor SB203580and non-competitive(allosteric type) p38inhibitors BIRB796, BIRB796might be more efficient inalleviating SM-induced cutaneous injury, which may because BIRB796simultaneously inhibited SM-induced activation of p38and JNK, while SB203580inhibited p38but enhance JNK activation.3Necroptosis inhibitors could prevent the cell death induced by SM, which indicatedthat necroptosis involves in the SM-induced cell death;4Among the drug combination regimen with anti-inflammatory drugs, capsaicinreceptor agonist olvanil in combination with PARP inhibitors MUS003showsignificantly protective effect against SM injury, which could be alternative drugcombination regimen for further research.In summary, Our study evaluated the protective effects of several kinds ofanti-inflammatory drugs based on different regulation mechanisms of inflammatoryresponse. We illustrated pharmacodynamic characteristics of many kinds ofanti-inflammatory drugs and got a optimal drugs combination regimen, which providesa reliable experimental basis for inflammation-based mechanism and drug research.. |
