The Prevention And Therapy Of Chixiaodou-Danggui-San On Skin Cancer

The increasingly serious environmental pollution and the aging population make the incidence of skin cancer in China rise year by year.It is generally believed that the occurrence of skin cancer is related to cumulative exposure to toxic substances or ultraviolet radiation.The major therapeutic means of skin cancer in modern medicine include surgical resection,chemoradiotherapy,which lead to a higher rate of recurrence and side effects,accompanied by the more treatment costs.Skin cancer belongs to "cauliflower-like sore"," malignant sore " and so on,which is also a type of abdominal mass and accumulation,and is treated according to sores and ulcers in traditional Chinese medicine.The common features in sore and ulcer are stagnation of qi and blood,stasis inducing fire syndrome and fire toxin injuring muscle.Chi Xiao Dou-Dang Gui-San(CDS)recorded by the Synopsis of Golden Chamber,has the functions of clearing heat and promoting diuresis,detoxify and drain pus,and removing blood stasis for promoting tissue regeneration,which is good for the sore pathogenesis in traditional Chinese medicine.The aim of this paper is to observe the preventive and therapeutic effects of CDS on skin cancer.This study was divided into three parts of in vivo,in vitro and mechanism.In vitro studies,A431 and B16-f10 cells were treated with different concentrations of alcohol extracts from CDS,and cell proliferation were detected by MTT;Laser holography was used to detect cell morphology and volume;The scratch was used to test cell migration.In vivo,firstly,the mouse skin cancer model induced by dimethylbenzanthracene was be established,at the same time,mice were gavaged with CDS.The tumor incidence rate and tumor nodule number on the back of experimental mice were recorded;The pathological changes of skin tissue on the back and liver tissue was observed by HE staining.The expression of EMT-related proteins in the skin tissues was detected by immunohistochemistry.Then,a melanoma allograft model was established,at the same time,mice were gavaged with CDS.The tumor size in tumor-bearing mice was recorded,the expression of EMTrelated proteins in tumor tissues was detected by immunohistochemistry.In order to study the mechanism,network pharmacology was used to predict the molecular mechanism of CDS in skin cancer prevention and treatment,and the mechanism was targeted at cell respiration,apoptosis,immune clearance and cell proliferation.In vitro,AO staining was used to detect the effect of CDS on autophagy in A431 and B16-f10 cells;The ROS and mitochondrial functions were tested by ROS kit and mitochondrial membrane potential kit in A431 and B16-f10 cells;The effect of CDS on Ras and Bcl-2 expression was detected in A431 and B16-f10 cells by immunofluorescence.In dimethylbenzanthracene induced skin cancer,ROS kit and mitochondrial membrane potential kit were used to detect the ROS and mitochondrial functions in skin tissues.The expressions of CD8,PD-1,TGF-?1 and VEGF in skin tissues were detected by immunohistochemistry;Immunofluorescence was used to detect the expressions of CD4,Caspas3,P16,Ras and Bcl-2 in skin tissues;Expressions of jak-2,Akt,Stata3,and AMPK in skin tissues was detected by Western blot.In the melanoma allograft model,ROS kit and mitochondrial membrane potential kit were used to detect the ROS and mitochondrial function of tumor tissues;Immunohistochemistry was used to detect the expressions of TGF-?1,PD-1,CD8 and VEGF in tumor tissues.Immunofluorescence was used to detect the expression of CD4,Caspas3,P16,Ras and Bcl-2 in tumor tissues;The expressions of Jak-2,Akt,Stata3 and AMPK?1 in tumor tissues were detected by Western blot.In vitro results showed that the alcohol extract from CDS at a certain concentration could change the cell morphology and inhibit cell proliferation and migration in A431 and B16-f10 cells.In vivo results showed that CDS could reduce the incidence of skin tumors and the number of tumor nodules in dimethylbenzanthracene-induced skin cancer-bearing mice,and also improve carcinogenic agent-caused skin and liver injury;Immunohistochemical results showed that the administration of CDS improved the EMT process of skin tumors on the back.In the melanoma allograft model,CDS decreased the growth rate of melanoma and improved the EMT process of tumor tissue.In the mechanism study,the network pharmacology analysis indicated that CDS can prevent skin cancer by regulating biological processes such as cell respiration,apoptosis,immune clearance and cell proliferation,as well as the signaling pathways such as AMPK pathways,PI3K-Akt and other signaling pathways.In vitro,CDS could improve the mitochondrial function in A431 and B16-f10 cells,reduce the level of ROS,promote autophagy,and reduce the expression of Ras and Bcl-2.In the mice skin cancer model induced by dimethylbenzanthracene,CDS could reduce ROS levels,improve mitochondrial function,reduce the TGF-?,VEGF,PD-1,Ras,Bcl-2,Akt,Stata3,Jak-2,AMPK?1 expression and increase the CD8,CD4,caspas3,P16 expression in skin tissues,which plays a role as maintaining skin tissue homeostasis.In the melanoma allograft model,CSD could reduce the ROS level,improve the mitochondrial function,reduce the expression of TGF-?,PD-1,VEGF,Ras,Bcl-2,Jak-2,Akt,stata3 and AMPK?1,increase the expression of CD8,CD4,caspas3,P16 in tumor tissues,which reduce the cancer cell malignancyIn summary,CDS can reduce cell malignancy to remove toxic sources by changing the cancer cell morphology,cell autophagic promotion and inhibition of cell proliferation and migration.At the same time,CDS can promote body rejection to maintain the skin cell homeostasis by regulating cell mitochondria function,signal transmission and system immunity,and therefore prevent skin cancer.