Study On Mechanism And Transdermal Delivery Of Parthenolide To Improve Psoriasis Like Skin Inflammation

Objective: Psoriasis is a chronic inflammatory skin disease,which is easy to relapse and is mainly mediated by immunity.There are currently no drugs that completely cure it,most only achieve clinical remission,and their pathogenesis is still being explored.Parthenolide(PTL),isolated from chamomile,is a sesquiterpenoid lactone that has shown potent antitumor,antiviral,and so on.This experiment investigated the ameliorative effects of parthenolide on psoriasis like skin inflammation based on IL-36 signaling,and parthenolide cream was prepared to evaluate its therapeutic effect for transdermal delivery.Methods: 1.BALB/c mice were used three times to establish an in vivo model of psoriasis like skin inflammation induced by imiquimod(IMQ).(1)The mice were randomly divided into seven groups: normal,model,PTL treated(4,2,1 mg/kg),vehicle alone(4 mg/kg),and dexamethasone(DXMS).Mice were gavaged with PTL(4,2,1 mg/kg)or applied DXMS ointment for the first 5days.(2)The mice were randomly divided into six groups: normal,model,PTL gavage treated(4mg/kg,i.g),PTL treatment by transdermal delivery(0.4,0.2 mg/ml,TTS),and DXMS.Mice were gavaged with PTL(4 mg/kg)or applied PTL cream(0.4,0.2 mg/ml)or DXMS ointment for the first 5 days.(3)The mice were randomly divided into four groups: normal,model,PTL treatment by transdermal delivery(0.4 mg/ml,TTS),blank cream treatment.Mice were applied PTL cream(0.4 mg/ml)or blank cream for the first 5 days.The topical dose of 62.5 mg of 5%IMQ cream was increased from day 6.The mice were sacrificed on day 10 for the collection of serum and dorsal skin.The psoriasis lesion area and severity index(PASI)was used to evaluate the back skin of mice.Histopathological and morphological changes were observed by H&E staining.Keratinocyte proliferation,epidermal differentiation,vascularity and inflammatory cell infiltration in the skin of mice were reflected by the expression of Ki67,Keratin1,Keratin14,Keratin16 and Keratin17 by immunohistochemical staining.The expression of macrophage marker F4/80,myeloperoxidase(MPO)released from neutrophils and neutrophil elastase(NE)was detected by immunofluorescence staining.The expression levels of inflammatory cytokines were determined by western blot,such as Il-36γ,Il-36α,Il-36β,Keratin16 and Keratin17.The m RNA levels of Il36 g,Il36a,Il36 b,Il1b and chemokines Cxcl1 and Cxcl2 were detected by real-time PCR(q PCR).2.Prescription design and preparation of PTL cream.The method for the measurement of parthenolide content by HPLC was developed to verify linearity,specificity,precision,repeatability,stability and sample recovery.3.Poly(I:C)was administered after pre-treatment of Ha Ca T keratinocyte with PTL(2,0.5,0.125 μM)and IL-36 receptor antagonist.The production and secretion of IL-36γ,IL-1β and caspase-1 in keratinocytes were determined by western blot.4.An ex vivo model of psoriasis was established by employing skin tissues of suckling rats.Poly(I:C)stimulation was given after pre-treatment of ex vivo tissue with PTL(2,0.5,0.125 μM)and IL-36 receptor antagonist,and culture fluid and skin tissue were recovered.The expression levels of IL-36γ,IL-1β,caspase-1,NE,Proteinase3(PR3),and Cathepsin G(CG)by western blot.Results: 1.PTL alleviated the phenomena of erythema,infiltration as well as thickening of the epidermis in a model of IMQ induced psoriasis like skin inflammation,reduced the expression of skin cell proliferation markers Ki67 and Keratin14,Keratin16,Keratin17.Keratin1 expression was elevated by PTL treatment.It also decreased the protein and m RNA expression of IL-36α,IL-36β,IL-36γ,and also exerted inhibitory effects on the m RNA levels of Il1 b and chemokines Cxcl1,Cxcl2.PTL was able to modulate the immune responses of macrophages and neutrophils by reducing the expression levels of F4/80,MPO,and NE.The improvement effect on psoriasis like skin inflammation was obviously enhanced by transdermal drug delivery treatment,in which blank cream also played an regulatory role.2.An HPLC content determination method for PTL was established.The chromatographic conditions were as follows: mobile phase consisted of water-acetonitrile,detection column temperature of 30°C,flow rate of 1.0 ml/min,wavelength at 230 nm,injection volume of 10 μl.The linearity of the PTL solution was good in the range of 1-400 μg/ml.The exclusive experiment showed that the retention time was 6.7 min,the blank matrix was not absorbed here,there was no interference in the determination of PTL,the individual peaks were well separated and the peak shape was symmetrical.The RSD for the precision experiment was 0.412%.The RSD of the repeatability experiment was 2.017%.The RSD for the stability experiment was0.303%.The drug recoveries were 100.27%,100.18%,100.84%,RSD were 2.24%,1.26%,1.73% respectively.The experimental results met the requirements specified in the methodology review.The mean drug content in the creams was 0.351mg/g,and the drug was evenly distributed in the creams.3.PTL can reduce IL-36γ induced by poly(I:C)stimulation in HaCaT keratinocyte,thereby inhibiting release of IL-1β and activation of caspase-1.4.Poly(I:C)stimulation not only promoted the production of IL-36γ,IL-1β and caspase-1in ex vivo psoriasis model,but also induced the expression of NE,PR3 and Cathepsin G,which were inhibited by PTL.Conclusion: Parthenolide can improve psoriasis like skin inflammation in vivo and in vitro,and may play a role by inhibiting IL-36 signaling.More significant effects were demonstrated after changing the mode of administration.It shows that PTL is a very potential drug for the treatment of psoriasis,and transdermal drug delivery also opens a new direction for further studies.