Phellodendron Charcoal And The Treatment Of Psoriasis

Looking at the records of charcoal medicine in ancient books,it can be seen that it has a wide range of clinical applications and about 100 types of diseases with multiple systems and multiple organs could be definitely ameliorated by charcoal medicine.As the new sources of the innovation of new drugs,exploring the undiscovered and brand-new effects of charcoal drugs,and scientifically explaining the material basis and mechanism,are of great significance for promoting charcoal drugs to serve the clinic with a wider range of bioactivities.Phellodendri Chinensis Cortex(PCC)was firstly accorded in "Shen Nong's MateriaMedica",and it is also one of the commonly used medicines in traditional prescriptions.Therefore,according to the previous results of the biological activities of some charcoal drugs,the effect of Phellodendri Chinensis Cortex Carbonisata(PCCC)on improvement the symptoms of imiquimod(IMQ)-induced psoriasis mouse model have been accidentally discovered.However,the basic scientific issues including the material basis and mechanism of PCCC have not yet been illustrated.To fill this research gap and prove the scientific rationality of PCCC in the treatment of psoriasis,the following series of studies have been carried out in this study.Objective:On basis of the new clue that PCCC possessed anti-psoriasis activity,this study confirm the effect of PCCC in the treatment of psoriasis by IMQ-induced psoriasis model,following by preliminarily explore the effect of the "fire" degree on its efficacy.Furthermore,combined with nanotechnology,chromatographic technology and optical technology,this study firstly proved that PCCC nano-components(PCCC-NCs)are the anti-psoriasis material basis of PCCC.Then the correlation between the physical and chemical properties of PCCC-NCs prepared with different processing parameters and its bioactivities were analyzed,following by screening the best preparing parameters of PCCC-NCs with anti-psoriasis.Based on this,the anti-psoriasis dose-effect relationship,administration route and biosafety of PCCC-NCs were further explored.The anti-psoriasis mechanism of this NCs was studied from the perspective of regulating the polarization of macrophages.This paper will provide leading research ideas for the clinical application and material basis research of traditional Chinese charcoal medicine.Method:(1)Using the IMQ-induced psoriasis model to evaluate the anti-psoriasis activity of PCCC with different administration routes and processing degree,and preliminary screen out the processing degree of PCCC,which lays the foundation for subsequent experiments.(2)The PCCC solution was separated into two parts by dialysis method,and the anti-psoriasis active part of PCCC was identified with the methods of chromatography,electron microscopy and optical technology.(3)The effects of carbonization temperature and time on the physicochemical properties of PCCC active parts were investigated,following by evaluating and optimizing the preparation parameters of it by IMQ-induced psoriasis model and Hacat cell proliferation model.Then the structural characteristics,element coordination and other information of PCCC active parts were analyzed by technologies applied in material domain.(4)The administration route and anti-psoriasis dose-effect relationship of PCCC-NCs were further studied by monitoring some indexes including body weight,spleen index,appearance of back and ear skin,PASI score,and pathological tissue changes(such as epidermal thickness,inflammatory cell infiltration and vascular proliferation,etc.).(5)The anti-psoriasis of immunohistochemical method mechanism were explored by detecting the level of M1/M2 marker factors in skin/serum,the ratio of Th1/Th2 and Th17/Treg in spleen cells,oxidation factors related factors as well as NF-?B/STAT6 pathway related protein expression using ELISA method,biochemical analysis method,flow cytometry technology,western blotting and immunohistochemical method,and explained from the perspective of regulating macrophages.(6)RAW264.7 cells were polarized into M1 and M2 subtypes by stimulated by lipopolysaccharide(LPS)and IL-4,respectively.The concentrations of active site of PCCC was selected on the based of the results obtained from CCK-8 experiment.After intervention of RAW264.7 cells by the active site of PCCC,the regulation effects of it on macrophages polarization were explored by detected the high expression factor of M1/M2 subtype macrophages.(7)The safety parameters of the active site of PCCC were obtained by cell CCK-8 experiment,acute toxicity and chronic toxicity experiment in mice.Result:(1)The anti-psoriasis effects of PCCC(PCCC sale in the market,PCCC-MS)was demonstrated in this study and the results showed that the activity of PCCC-MS by intraperitoneal injection was better than that of intragastric administration and subcutaneous injection.Additionally,the anti-psoriasis effect of processing products of Phellodendri Chinensis Cortex(PCC)with different processing degrees were compared by IMQ-induced psoriasis model,and the results depicted that the increased carbonized degree of PCC's processing products may lead to the enhancement of its therapeutic activity for psoriasis.The detailed order of anti-psoriasis activity from strong to weak was listed as followed:PCCC prepared at 400?(PCCC-400?)>commercial PCCC(PCCC sale in the market,PCCC-MS)>Fried PCC(F-PCC)>PCC.(2)Results showed that the components inside dialysis bag(MWCO>1000)was the active part of PCCC-400? performed anti-psoriasis effect and HPLC profiles of it appears no absorption peak.Spherical and size distribution at the ranges of 0.5?3.6 nm particles with obvious lattice(0.22 nm)could been seen in low resolution transmission electron microscopy(TEM)and high resolution TEM(HRTEM).Infrared and fluorescence spectra showed that active part of PCCC-400? possessed fluorescence,and abundant functional groups such as hydroxyl and carboxyl groups on the surface.Additionally,the similar analysis results were presented in the components inside dialysis bag of PCCC-MS.Combined with the research results of our team,this study confirmed that the anti-psoriasis activity material basis of PCCC is nano-components(NCs),which has been named as PCCC-NCs.(3)The HPLC profile showed that there were no small molecule compounds in PCCC-NCs prepared under nine kinds of preparing conditions,and the physical and chemical properties of them were further analyzed by technologies applied in material domain.It could be seen that when the carbonization temperature was the same,the particle size of PCCC-NCs decreased and the fluorescence maximum excitation wavelength showed a blue shift with the preparation time prolonging.Additionally,when the carbonization time is the same,temperature increasing will also cause the decreasing tendency of particle size of PCCC-NCs,but the fluorescence wavelength changes were not regular.In addition,the preparing temperature and time of PCCC-NCs could also cause the changes in infrared absorption peak intensity and lattice spacing,but the regularity with the current results was difficult to explain.Furthermore,guided by the in vitro and in vivo anti-psoriasis efficacy,the optimal preparation parameters for obtaining PCCC-NCs were optimized at 400?,1h.The detailed characterization results presented that PCCC-NCs were mainly composed of carbon and oxygen elements,and existed highly amorphous carbon structure and ?-?*,n-?*transitions,as well as fluorescence excitation-emission dependence.(4)Based on the above results,this study further proved that PCCC-NCs obtained at 400?and 1 hour treated by intraperitoneal injection exhibited better curative effect on alleviating PASI score of psoriasis mice than intragastric administration and subcutaneous injection.Additionally,the high,medium and low doses of PCCC-NCs showed remarkable effects on improving the weight loss,elevated spleen index,PASI score,back and ear skin pathological changes(such as epidermal thickening,inflammatory cell infiltration,and vascular proliferation)caused by IMQ intervention.The changes in related indicators or protein expression in skin tissues further verified above-mentioned results.Comprehensive analysis found that the anti-psoriatic dose-effect curve of PCCC-NCs showed a "U" shape,which suggested that,middle-dose PCCC-NCs showed a better therapeutic effect within the experimentally investigated doses.(5)Mechanism studies showed that PCCC-NCs could reduce the M1-related cytokines both in serum(TNF-?,IL-6,IL-23,IL-17A and IL-22)and skin tissues(TNF-?,IL-6,NO and iNOs)in IMQ-induced psoriasis mouse,and skin immunohis-tochemical analysis shows that PCCC-NCs can reduce the positive expression of CD86,indicating that PCCC-NCs could inhibit the M1-type macrophages polarization.Additionally,the levels of M2-related cytokines(IL-10 and Arg-1)in the serum and skin tissues could be elevate by PCCC-NCs,indicating that it possessed the ability for promoting the macrophages polarization to the M2 subtype.The Flow cytometry results exhibited that PCCC-NCs could regulate T cell balance by reducing Th1/Th2 and Th17/Treg,and the decreased T-AOC,SOD,GSH-Px,CAT and elevated MDA induced by IMQ could be improved by PCC-NCs.Further research found that PCCC-NCs could inhibit the phosphorylation level of p65 and promote the phosphorylation level of STAT6 at the same time.These above-mentioned results indicated that PCCC-NCs could improve the elevated M1/M2 of macrophages caused by IMQ by regulating the NF-?B and STAT6 pathways,following by reducing the inflammatory mediators secreted by M1 macrophages and promoting the levels of anti-inflammation mediators secreted by M2 macrophages.Then the imbalance of Th1/Th2 and Th17/Treg,and oxidative damage were further relived.(6)PCCC-NCs could alleviate the M1 polarization of RAW264.7 macrophages induced by LPS,and promote the M2 polarization induced by IL-4.These results further confirmed the effect of NCs on regulation macrophage polarization function in vitro perspective.(7)The safety evaluation results showed that the safe doses of PCCC-NCs on L02 and 293T cells was ?1250 ?g/mL.and NCs concentrations ranging from 1.56 mg/kg?100.00 mg/kg cause no acute toxicity to mice.In addition,the animal's physiological state and major organs show no significantly changes when the maximum dose reached 200 mg/kg.In the long-toxicity study,animal's physiological state,body weight,food intake,blood routine,serum biochemical indicators,main organ coefficients and pathological tissues showed no obvious changes after treating with PCCC-NCs for 1 month and discontinued for 2 weeks.Conclusion:This study firstly proved that PCCC possessed anti-psoriasis activity from a pharmacological point of view,and it with a high carbonization degree exhibited a better effect by intraperitoneal injection.Comprehensive using of chromatographic technology and material science-related technology,and traditional Chinese medicine pharmacology and efficacy research methods,tracking and confirming that the anti-psoriasis material basis of PCCC was PCCC-NCs.Then using this component as a starting point,the preparation parameters of PCCC was optimized and standardized by using anti-psoriasis activity of PCCC-NCs as elevating index,and the best preparation parameters of the NCs was 400?,1h.The anti-psoriasis dose-effect relationship of PCCC-NCs was not in dose-dependent manner and it could exert the anti-psoriasis activity by regulating the macrophages polarization through the NF-?B and STAT6 pathways,following by improving the imbalance of T cell and oxidative damage.In addition,PCCC-NC s showed extremely high safety in cells toxicity assay,acute toxicity and long-toxicity experiments in mice.These results present in this paper confirm that PCCC possessed anti-psoriasis activity,and expounding its scientificity from the perspective of material basis and mechanism.It will not only provide exemplary ideas for the promotion of basic research on the innovation efficacy,material basis and specifications and optimization technologies of traditional Chinese medicine charcoal drugs represented by PCCC,but also hold a promising potential for PCCC-NCs to be a viable alternative to current strategies for psoriasis treatment.