Efficacy And Mechanism Of Chlorophyll Dihydroporphyrin Derivative Photodynamic Therapy For Acne

Background:Acne vulgaris is a chronic inflammatory skin disease involving the pilosebaceous unit.Eighty-five percent to ninety percent of the population have varying degrees of acne,twenty percent of which undergo severe acne.Severe acne is difficult to heal and easy to leave permanent scars,which seriously affects the appearance and brings serious impact to the physical and mental health of patients with acne.At present,the traditional treatment of severe acne is mainly oral isotretinoin and tetracycline antibiotics.The course of those traditional treatments last for several months,which can often leave damage of liver and kidney,gastrointestinal reactions,teratogenicity,affect bone development and induce or aggravate depression.Risks restrict its clinical application to a certain extent.5-aminolevulinic acid photodynamic therapy(ALA-PDT)is a new method of drug-mechanical combination,which can damage specific lesions and proliferative tissues by generating reactive oxygen species(ROS).In recent years,a large number of clinical studies at home and abroad have confirmed that ALA-PDT is safe and effective in the treatment of severe acne.Our research group has carried out PDT for acne for more than 10 years.We have determined the best PDT treatment parameters for Chinese acne patients and successfully treated thousands of patients with severe acne,which further formulated the clinical application guidelines of ALA-PDT in China.ALA-PDT is considered to be the most effective phototherapy for severe acne.Pain,erythema,dry skin,edema,burning sensation,pustules,blisters,and even exudation,itching,and hyperpigmentation often occur during ALA-PDT treatment of acne.And the incidence of pain,erythema,burning sensation,and dry skin is far higher than itching,pustules and crusting.Especially for teenagers,severe reactive acne often occurs in a short period of time after PDT.Secondly,ALA is a prodrug of the photosensitizer,which itself does not have photosensitizing effect.After administration,it needs time for converting into a photosensitizer before it can play a photosensitizing effect.Expensive,which limits its clinical application to a certain extent,so the exploration of safe and efficient new photosensitizers is an important breakthrough in research.Chlorophyll-based photosensitizer PDT can effectively inhibit seborrhea,treat acne and large pores.And the application time of the chlorophyll-based photosensitizer is only half an hour,and there are no adverse reactions such as redness,swelling and pain during and after treatment.However,there is no relevant research on the treatment of acne with chlorophyll-based derivatives,and there is also a lack of specific parameters and mechanisms of the chlorophyll-based derivatives Shengtaibufen photodynamic therapy(STBF-PDT).Ferroptosis is a new type of iron-dependent programmed cell death with specific morphological and biochemical characteristics,which mainly iron overload and ROS-dependent glutathione peroxidase 4(GPX4)depletion caused by the accumulation of lipid peroxides.Whether STBF-PDT can inhibit lipid secretion by inducing ferroptosis in sebocytes.At the same time,previous studies have shown that mammalian target of rapamycin(mTOR)can inhibit ferroptosis by promoting the expression of GPX4,and inhibiting the expression of mTOR can further inhibit GPX4,thereby promoting the occurrence of ferroptosis.PDT induces ferroptosis by inhibiting the expression of mTOR.In addition,PDT can also inhibit sebum secretion by inhibiting mTOR and sterol-regulatory element binding proteins 1(SREBP1).However,whether STBF-PDT induces sebocyte ferroptosis and inhibits sebum secretion by inhibiting mTOR/GPX4 and mTOR/SREBP1 remains to be further confirmed in this study.Objectives:This topic intends to carry out research around the above problems to achieve the following research objectives:(1)To explore the kinetic parameters of STBF on the skin surface;(2)To clarify the efficacy and safety of STBF-PDT for acne;(3)To explore the effect of STBF-PDT on sebaceous gland plaques in golden hamster;(4)To study whether STBF-PDT induce ferroptosis to inhibit sebocyte proliferation and sebum secretion via mTOR/GPX4 and mTOR/SREBP1 pathways.Methods:1.Exploring STBF fluorescence kinetic parameters:the absorption wavelength of STBF was determined by spectrophotometer and the fluorescence was photographed after UV light irradiation;STBF was applied for 0.5h,1h,2h,3h,4h,5h,and the fluorescence intensity on the forearm skin was detected by fluorescence spectrometer.The fluorescence intensity of different concentrations of STBF(1mg/ml,0.5mg/ml,0.25mg/ml and 0.125mg/ml)after external application on the facial skin of acne patients.2.Randomized single-blind split face controlled study to evaluate the efficacy and safety of STBF-PDT in the treatment of acne:The face of acne patients was randomly divided into left and right halves.The optimal concentration of STBF solution was externally applied on one side of the face for 0.5h,and the other side was externally applied with matrix solution for 0.5h.And then the whole face was irradiated with LED red light(energy density 150J/cm2,power density 100mW/cm2).The efficacy of STBF-PDT in the treatment of acne was evaluated by measuring the lesion clearance rate,the number of fluorescent spots on the face under VISIA ultraviolet light and the physiological parameters of the skin.3.To evaluate the therapeutic effect of STBF-PDT on sebaceous plaques in golden hamsters:Golden hamster sebaceous plaques were randomly divided into control side and treatment side.STBF-PDT treated golden hamster sebaceous plaques.Fluorescence frozen section,HE staining,OCT detection,Nile red staining and Western Blot were used to evaluate the therapeutic effect of STBF-PDT.4.To explore the inhibitory effect of STBF-PDT on sebaceous gland cells:Fluorescent enzyme labeling instrument was used to detect the fluorescence intensity of STBF after application to determine the optimal application concentration;Cells were divided into control group,single-light group(light group),single-drug group(STBF group),low dose STBF-PDT group(0.2J/cm2 PDT group),medium dose STBF-PDT group(0.4J/cm2 PDT group)and high dose STBF-PDT group(0.8J/cm2 PDT group).The survival rate of sebaceous gland cells was assessed by CCK8,the generation of ROS was detected by DCFH-DA fluorescent probe,and the secretion of sebum was detected by Nile red staining.5.Defining STBF-PDT-induced ferroptosis in sebaceous glands:Detecting structural changes in sebaceous glands treated with STBF-PDT by transmission electron microscopy.The unstable iron in the cytoplasm after STBF-PDT was detected by ferrous ion staining.Sebum secretion was observed by Nile red staining.The expression of ferroptosis-related proteins was detected by Western Blot.6.To clarify the mechanism of STBF-PDT-induced ferroptosis in sebocytes:after treatment with ferroptosis promoter RSL3 and ferroptosis inhibitor Fer-1 combined with STBF-PDT,CCK8 was used to evaluate the survival of sebaceous gland cells,and ferrous ion staining was used to detect the situation of unstable iron ions in the cytoplasm after STBF-PDT.Sebum secretion was observed by Nile red staining.The expression of ferroptosis-related proteins was detected by Western Blot.Results:1.Determination the fluorescence kinetic parameters of STBFSTBF UV fluorescence spectrophotometer detected strong absorption peaks at 401.9nm,504.9nm and 655.0nm,and showed red fluorescence under UV light irradiation.The results show that the best application of STBF in clinical treatment is 0.5mg/ml,and the application time is 0.5h.2.Randomized single-blind split face controlled study to evaluate the efficacy and safety of STBF-PDT in the treatment of acne22 patients with acne were recruited.And 20 of them completed all sessions of treatment.And the clearance rate of total lesions was 67.42±8.51%in the STBF-PDT group and 41.05±11.97%in the control group 4 weeks after the treatment.There was a statistically significant difference between the two groups(P<0.001).The clearance rate of non-inflammatory lesions was 17.13±14.48%in the STBF-PDT group and 16.33±11.52%in the control group,with no significant difference between the two groups(P≥0.05).The average clearance rate of inflammatory lesions was 84.41±7.13%in the STBF-PDT group and 50.10±13.91%in the control group,with a statistically significance(P<0.0001).The skin sebum of the STBF-PDT treated side was significantly lower than that on the control side.3.STBF-PDT can effectively reduce sebaceous plaques of golden hamstersAfter STBF-PDT treatment,HE and OCT both confirmed that the sebaceous plaques of golden hamsters were significantly reduced.Nile red staining showed the sebum secretion decreased.4.Confirm the inhibitory effect of STBF-PDT on sebocytesThe optimum concentration of STBF-PDT to treat sebocytes was determined to be 2.5μM and the application time was 1h.At 0.4J/cm2,the inhibition rate of STBF-PDT on sebocytes was 50%.STBF can enter the cytoplasm of sebocytes after incubation.STBF-PDT can induce a large amount of ROS and significantly reduce sebum secretion.5.Confirm that STBF-PDT induced ferroptosis in sebocytesElectron microscopy results showed that the mitochondrial cristae of sebocytes were reduced or even disappeared after STBF-PDT and the outer membrane was broken.In the STBF-PDT group,the production of ferrous ions increased significantly and the secretion of sebum decreased.6.Confirm that STBF-PDT inhibits sebocyte proliferation and sebum secretion by inhibiting mTOR/GPX4 and mTOR/SREBP1 pathwaysThe expressions of pmTOR,pS6K,GPX4 and SREBP1 in STBF-PDT group were significantly down-regulated.After promoting ferroptosis,the production of ferrous ions was more,the secretion of sebum was less,and the down-regulation of pmTOR,pS6K,GPX4 and SREBP1 was more obvious.After inhibiting of ferroptosis,ferrous ion production was reduced,sebum secretion was restored,and pmTOR,pS6K,GPX4,and SREBP1 were significantly up-regulated.Conclusions:1.STBF-PDT can effectively and safely be used for the treatment for acne and can significantly inhibit sebum secretion.2.STBF-PDT can reduce the sebaceous gland plaques of golden hamsters and reduce the sebum of sebaceous gland plaques.3.STBF-PDT can enhance ROS,induce ferroptosis in sebocytes and inhibit the proliferation and sebum secretion of sebocytes.4.STBF-PDT induced ferroptosis and inhibits sebum secretion via mTOR/GPX4 and mTOR/SREBP1 pathways.