The Role Of Drp1 In Regulating Mitochondrial Fission Influences Pyroptosis In Atopic Dermatitis

Objective :Atopic dermatitis(AD)is a chronic inflammatory cutaneous disorder characterized by recurrent skin lesions and intense pruritus,which can affect patient's sleep,quality of life,physical and mental health.The pathogenesis and etiology of AD are complex and not yet completely understood.Skin barrier dysfunction,genetic factors,immune disorders and environmental factors are all related to the pathogenesis of AD.For the treatment of AD,in recent years,with the application of biologicals represented by Dupilumab,more effective methods have been found for the treatment of AD patients.But at present,the recurrence and obstinacy of AD are still difficult problems frequently encountered in clinical practice.Therefore,further research on the pathogenesis of AD can provide new ideas for clinical treatment.Atopic dermatitis is characterized by complex immune mediated inflammation which caused by dysfunctional epidermal barrier and penetration of microbes or allergens.It is reported that inflammasomes which are multiprotein complexes assembled by intracellular NOD-like receptors play a major role in pathogen recognition and inflammation.Accumulating evidences now show that inflammasomes are involved in the inflammatory response in AD.Among them,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome has attracted more attention.In general,NLRP3 inflammasome activation is activated through TLR/NF-?B signaling pathway,then pro-caspase-1 is activated,activated caspase-1 rapidly cleaves the proinflammatory cytokines IL-1? and IL-18,results in their maturation and release into extracellular,inducing a strong immune response.In addition,activated caspase-1can cleave Gasdermin D(GSDMD)to release its N-terminal domain to form holes on the cell membrane inducing pyroptosis.Pyroptosis is a pro-inflammatory programmed cell death mediated by inflammasomes activation.There has demonstrated that activating NLRP3 inflammasome in keratinocytes,consequent release of IL-1 proteins might be a risk factor for AD,suggesting that NLRP3 inflammasome-mediated pyroptosis plays a role in the pathogenesis of AD.Therefore,inhibition of NLRP3 inflammasome may be a new treatment for AD.Dynamin-related protein 1(Drp1)-a member of the dynamin protein family,is a important protein regulating mitochondrial fission.In response to cell stress,cytoplasmic Drp1 is transferred to mitochondria and promotes mitochondrial fission,participating in the occurrence and development of mitochondrial dysfunction.Mitochondria form dynamic networks governed by a balance between fission and fusion processes,maintaining the normal shape and function of mitochondria.Imbalance of fission and fusion causes mitochondrial dysfunction.It has been revealed that excessive mitochondrial fission is associated with numerous pathologies,such as neurodegeneration,liver ischaemia-reperfusion injury and inflammatory airway diseases.Moreover,Drp1 activation-induced mitochondrial fission causing NLRP3inflammasome-related pyroptosis in cardiomyocytes via caspase-1 dependent manner,aggravates myocardial injuries;maintaining mitochondrial dynamics can reduce NLRP3 related pyroptosis to exert an anti-atherosclerotic effect.These studies suggest that Drp1 is involved in the regulation of NLRP3 inflammasome mediated pyroptosis.However,whether Drp1 is involved in NLRP3 inflammasome mediated pyroptosis in the pathogenesis of AD remains unclear.At present only studies have shown that compared with healthy controls the expression of Drp1 in the skin of AD patients was significantly increased,inhibition of Drp1 activity or down-regulation of Drp1 expression can inhibit mitochondrial translocation,degranulation and tumor necrosis factor secretion of mast cells.Therefore,this study conducts in cell and animal experiments to study the effects of Drp1 inhibition on mitochondrial function?NLRP3activation and pyroptosis,further elucidates the pathogenesis of AD,clarifies whether Drp1 can be used as a clinical treatment target,and provides new ideas for clinical drug development.Methods : The human epidermal keratinocytes(HEKs)were treated with AD-like inflammatory cocktail [polyinosinic–poly-cytidylic acid(10 ng/ml),TNF-?(10 ng/ml),IL-4(50 ng/ml),IL-13(50 ng/ml)],Mdivi-1-a selective inhibitor of Drp1 was performed,Drp1? p-Drp1?NLRP3?ASC? Gasdermin D-NT? Caspase-1 and IL-1? were detected by Western blot,the lactate dehydrogenase(LDH)level was detected by visible spectrophotometry,NF-?B signaling activity was determined by Western blot and immunofluorescence,ROS release was detected by flow cytometer,Western blot was used to detect the expression of mitofusin1(Mfn1),mitofusin2(Mfn2)and optic atrophy1(Opa1);si RNAs against human Drp1 were transfected into keratinocytes,accompanied by treated with AD-like inflammatory cocktail,the expression of Drp1 was detected by RT-q PCR,and the expression of Drp1?NLRP3?ASC and Gasdermin D-NT after transfection was detected by Western blot,through the above studies we could preliminarily define the effect of Drp1 inhibition on NLRP3 inflammasome activation and pyroptosis in AD-like inflammatory cocktail induced HEKs,and identify the related signaling pathways.Then to clarify the therapeutic effect of Drp1 inhibition on AD,the AD mouse model was induced by 2,4-dinitrochlorobenzene(DNCB),and intraperitoneal injection of Mdivi-1 was performed.At the end of the experiment,the times of scratching of the mice were observed,dermatitis score was performed,Ig E level and AD related inflammatory factors level(IL-4,IL-5 and IL-13)were detected by ELISA.The pathological change of skin lesion was detected by TB staining and the infiltration of mast cells was detected by TB staining.Meanwhile to elucidate the mechanism of Drp1 inhibition on AD,in DNCB induced AD mouse model,the expression of Drp1 was detected by RT-q PCR,the expression changes of Drp1 ? p-Drp1 ? NLRP3/pyroptosis related indexes were detected by Western blot,LDH level was detected by visible spectrophotometry,the activity of NF-?B signal was detected by Western blot and immunofluorescence,the expression of Mfn1,Mfn2 and Opa1 were detected by Western blot.Results :1.The expression of p-Drp1 was increased in keratinocytes stimulated by AD-like inflammatory cocktail.2.The Drp1 selective inhibitor Mdivi-1 could inhibite the expression of NLRP3?ASC? cleavage of caspase-1?GSDMD-NT? IL-1? and IL-18,decrease LDH level,decrease phosphorylation of P65 and I?B?,prevent nucleation of P65,elevate the expression of mitochondrial fusion proteins Mfn1?Mfn2 and Opa1 and decrease ROS release in keratinocytes under AD-like inflammatory.3.After transfection of si RNA targeting Drp1 into keratinocytes,the expression of Drp1 was decreased.Knocking down Drp1 decreased LDH level and the expression of NLRP3?ASC?GSDMD-NT in keratinocytes stimulated by AD-like inflammatory cocktail,the data suggested that Mdivi-1 could exert same function as Drp1 RNA silencing in keratinocytes stimulated by AD-like inflammatory cocktail.4.In DNCBinduced AD mice,Mdivi-1 treatment significantly attenuated AD-like symptoms of mice,lowered serum Ig E level,reduced the production of IL-4 ? IL-5 and IL-13,ameliorated the thickening of epidermis and decreased the number of mast cells and eosnophils in skin tissues from AD mice.5.In lesional skin of DNCB-induced AD mice,the Drp1 inhibitor Mdivi-1 could reduce the expression of pyroptosis associated indexes NLRP3? ASC? cleaved caspase-1? GSDMD-NT? IL-1? and IL-18,decrease LDH level,decrease phosphorylation of P65 and I?B?,prevent nucleation of P65 and increase the expression of mitochondrial fusion proteins Mfn1?Mfn2 and Opa1.Conclusion: In cell and animal AD models,inhibition of Drp1 protects against experimental AD through inhibiting mitochondrial fission and subsequently blocking NF-?B signal pathway,then inhibiting the activation of NLRP3 inflammasome and reducing pyroptosis.Drp1 inhibitor Mdivi-1 may be a new drug option for the treatment of AD.