The Effect And Mechanism Of Chinese Herbal Medicine Rosin And It's Bioactive Constituents Against Psoriasis-like Dermatitis

Rosin(also known as colophony)is an exudate of conifer trees such as Pinus masscnlana(Pinaceae)and has a wide range of biological activity and industrial applications.It has always been regarded as an important medicinal resource in traditional Chinese medicine,and the processed rosin has been used to treat skin diseases such as tinea,scabies and wounding for more than 2,000 years.It has also been used to treat psoriasis in folk and some hospital of Chinese medicine for its notable efficacy.Unfortunately,the side-effects caused by improper processing of rosin are also noticeable,and the mechanism of its anti-psoriasis effect is still unclear.Objective:Based on the ancient literatures of rosin,we used animal models of psoriasis-like dermatoses induced by imiquimod(IMQ)to verify the in vivo biological activity of water-boiled rosin(WBR)in the treatment of psoriasis and to elucidate its potential mechanism.The main constitutions of rosin were studied through the separation and structure determination.The content change of the main component abietic acid(AA)in WBR was measured by high performance liquid chromatography(HPLC).Next,the anti-psoriasis effect of AA,the main component of rosin,was also observed,and its potential mechanism for the treatment of psoriasis-like dermatitis through modulation of gut microbiota was investigated.These studies will provide experimental evidences for further clinical application of rosin and AA in the treatment of psoriasis or anti-inflammatory therapyMethods:1.By reviewing the ancient literatures,the evolution of rosin aliases,origin,nature,taste,meridian,efficacy,taboos,toxic and side effects were systematically summarized.2.The model mice with imiquimod(IMQ)-induced psoriasis-like inflammatory were used to observe the anti-psoriasis efficacy of WBR in vivo and explore its anti-inflammatory mechanism.3.The purified resin acids were separated from WBR using normal column chromatography,medium pressure preparative chromatography(MPLC)and HPLC.The mass spectrometry(MS),various chromatographic,and spectral techniques were used to identify the isolated pure compounds from WBR.Finally,the content of AA in natural rosin and WBR was analyzed using HPLC.4.Using IMQ-induced psoriasis-like inflammatory mouse model,we verify the efficacy of AA(the main ingredient of rosin)against psoriasis,and test its inhibitory bioactivities on psoriasis-related inflammation.Meanwhile,we detected gut microbiota reshaped by AA using 16S rRNA sequencing technology.Finally,single and fecal bacteria transplantation experiments were used to explore the potential mechanism of AA against psoriasis-like dermatitis through regulation of the gut microbiota.Results:Textual research showed that rosin was first recorded in Divine Farmer's Classic of Materia Medica,and its name was songzhi(rosin gum).The name of songxiang(rosin)was first recorded in Materia Medica of Diannan or Complete Record of Benevolence.In the anicent literatures,pine trees used to obtain rosin are widely distributed in China.In historical records,the origin,nature,taste,meridian,processing,taboos,and toxic and side effects of rosin are consistent with modern literatures,but not the efficacy and applications.Due to the historical changes,the clinical application of rosin has been gradually limited to the treatment of surgical and skin diseases.2.WBR significantly alleviated the PASI score of skin lesions of IMQ-induced psoriasis-like inflammation in mice(P<0.01),while inhibiting the expression of K17 and PCNA in the skin of mice(P<0.01),down-regulating the percent of Thl/Th17 in mouse spleen(P<0.05),in addition,decreasing the relative expression of IL-17A,IL-17F,IL-22,IL-23,and TNF-? in mouse skin(P<0.05 or P<0.01)3.To clarify the bioactive constituents of WBR against psoriasis-like dermatitis,we separated the main compounds from WBR to be rosin acid including AA,neoabietic acid and palustric acid.By measuring the content of AA in in natural rosin and WBR,we found that the content of AA in natural rosin was increased from 13.96%(w/w)to 29.90%(w/w)after boiling4.1)Oral administration of AA can significantly alleviate IMQ-induced psoriasis-like inflammation in mice and reduce the PASI score of skin lesions(P<0.01).Histopathology showed that AA significantly reduced IMQ-induced skin epidermal thickness,hyperkeratosis,parakeratosis,acanthosis,perivascular infiltration of inflammatory cells,and the expression of PCNA and K17.Meanwhile,compared with model group mice,the proportion of Thl7 cells and spleen Treg cells(CD4/CD25/Foxp3)were significantly decreased in AA group(P<0.05),and the relative expression of serum IL-17A?IL-23?TNF-?,and IL-1? was lower(P<0.05).2)Fecal microbiota 16S rRNA sequencing analysis showed that,compared with IMQ group,the relative abundance of Firmicutes and Tenericutes at phylum level was decreased respectively in AA group,whereas the relative abundance of Bacteroidetes was increased in AA group(P<0.05).At class level,Bacilli were increased in AA group compared with IMQ group(P<0.05).At order level,Bacillales were increased in AA group(P<0.05).At family level,AA can enhance the relative abundance of Planococcaceae?Porphyromonadaceae?Enterobacteriaceae and Prevotellaceae compared with that of model mice(P<0.05 or P<0.01).Finally,the relative abundance of Anaerotruncus and Christensenella at genus level was tested to be increased in AA group,whereas the relative abundance of Kurthia,Citrobacter and Klebsiella increased in AA group compared with IMQ-treated group(P<0.05 or P<0,01).3)Transplantation of Kurthia massiliensis cannot alleviate IMQ-induced psoriasis-like dermatitis in mice.4)Finally,fecal microbiota transplantation(FMT)showed that the fecal microbiota from AA-treated mice can alleviate IMQ-induced psoriasis-like inflammation in mice,and the PASI score of skin lesions and inflammatory factors in FMT group were significantly decreased compared with those in IMQ group and inactivated fecal bacteria transplantation(IFMT)group(P<0.01 or P<0.05).High-throughput 16S rRNA sequencing showed that,oral administration of fecal microbiota cannot reshape the same gut microbiota that induced by AA.Comparing with PBS and/or IFMT,the abundances of gene Erysipelotrichaceae incertae_sedis and Intestinimonas in FMT group were increased,whereas the abundance of gene Anaerotruncus was decreased(P<0.01 or P<0.05).Conclusions:1)Textual research showed that rosin was first recorded in Divine Farmer 's Classic of Materia Medica,and its name was songzhi(rosin gum).The name of songxiang(rosin)was first recorded in Materia Medica of Diannan or Complete Record of Benevolence.In the ancient literatures,the origin,nature,taste,meridian,efficacy,taboos,and toxic,side effects,and processing methods of rosin are more consistent with modern literatures,but its efficacy and application vary.With the change of history,the clinical application of rosin is gradually limited to the treatment of surgical and skin diseases in traditional Chinese medicine.According to modern researches,rosin acids in rosin have a variety of biological activities,such as anti-tumor,anti-inflammatory and so on,which suggests that rosin still has many potential efficacy to treat more disorders.2)Using an IMQ-induced psoriasis-like inflammatory mouse model,we found WBR is effective for the IMQ-induced psoriasis-like inflammation with the efficacy arising from its proliferation inhibition of Thl/Th17 cells and epidermal keratinocytes via the downregulation of the relevant inflammatory cytokines such as IL-23,IL-17A,and IL-17F in IL-23/IL-17 immune axis,and WBR is no toxicity,which suggested that water-boiling method is important for reducing the toxicity of rosin.3)The main components of WBR were found to be AA,neoabietic acid,and palustric acid.And the content of AA in natural rosin and WBR were measured,finding that the content of AA in water-boiled rosin was higher obviously than that in natural rosin.4)Oral administration of AA can also alleviate IMQ-induced psoriasis-like inflammation in mice by modulating the immune response associated with Th17 cells.Those results suggested that water-boiling method can improve its pharmaceutical effect by enhancing the content of AA in rosin.This finding also illustrated that ameliorating psoriasis-like inflammation effect of AA might depend on gut microbiota,but altered Kurthia massiliensis alone cannot replace the regulation of AA on gut microbiota.