Based On The FASN Target To Study The Mechanism Of The Effective Monomer Berberine Of Xianglian Pills In Inhibiting Human Colon Cancer Cells

Objective It is clear that FASN and ACC is the key target of colon cancer,and the single traditional Chinese medicine with the highest occurrence frequency and the drug pair with the highest compatibility frequency in Xianglian pill in the past dynasties are explored to verify that the effective monomer berberine of Xianglian pill can play an anti-tumor role by regulating ACC/FASN lipid metabolism-related signal pathway.Methods1.Literature mining and bioinformatics(1)Literature mining: all the prescriptions named Xianglian Wan in the Northern Song Dynasty and contemporary times were searched in the medical encyclopedia website,and the traditional Chinese medicine composition of Xianglian Wan in the past dynasties was input into the traditional Chinese medicine inheritance assistant system V2.5 to analyze the traditional Chinese medicine that appeared the most frequently and its matching.(2)Clinical bioinformatics:(1)In the ETCM database,click on the Formulas module to retrieve "Xiang Lian Wan",showing the gene enrichment analysis of Xianglian pills target components.The FDR value was sorted from small to large,and the GO term with significant difference among the TOP 20 was selected.The original data was imported into the We Chat platform to generate the enrichment bubble diagram.(2)In the GEPIA2 database,A."Expression Analysis: Expression DIY";B."Box plot";C."Gene: FASN";D."log2FC | Cutoff: 2,p value the Cut off: 0.001";E."Cancer name:COAD";F."Match TCGA Normal and GTEX Data".(3)In the GEPIA2 database,A."Expression Analysis: General";A.Quick Search B.Quick Search C.A.Exploration;B.B.Gen＿DE;C.Gene Expression: FASN.(4)In the TIMER2.0 database: a.B.Gen＿DE;C.Gene Expression: FASN.(5)In the TCGA database,the relationship between Fatty acid synthetase(FASN)and survival time,as well as the relationship between FASN and BMI in colon cancer patients were analyzed by R language.The relationship between FASN and survival in obese colon cancer patients was analyzed by R language.(3)Structural information biology: CB-Dock website completes molecular docking.2.Animal experiments(1)Modeling intervention and administration: Subcutaneous tumor formation in nude mice was firstly induced by subcutaneous injection of Lovo cells,and then subcutaneous tumor was inoculated in situ to establish the model of in situ colon cancer.Hyperlipidemia model was established in nude mice on high PA diet.They were divided into blank group,in situ tumor group,PA group,Xianglian pill +PA group,berberine(low)+PA group,berberine(medium)+PA group,berberine(high)+PA group,orlistat +PA group,a total of 8 groups,6 mouses in each group,were treated for 28 days.(2)Detection items:(1)Weighing the mass of the tumor and the weight of the tumor.(2)Acetyl CoA,NADPH,Malonyl CoA in the serum of nude mice were detected by ELISA,and four items of serum lipids were detected by biochemical assay.(3)The expression and distribution of FASN and ACC proteins in tumor tissues and intestinal tissues of nude mice were detected by immunohistochemistry.(3)Statistical method: One-way analysis of variance was used.3.Cell experiment:(1)Different doses of berberine(0 μM,25 μM,50 μM,100 μM,150 μM,200 μM)were used to intervene SW480 colon cancer cells for 24 h,48 h,72 h,and CCK8 method was used to screen the effective action time and concentration of berberine in SW480 cells.(2)The influence of berberine and palmitic acid on FASN protein expression was observed by Western Blot.Results1.Literature mining and bioinformatics(1)Literature mining: Through the analysis of the prescription named Xianglian pill in the past dynasties,the Chinese medicine with the most frequent occurrence was Rhizoma Coptis(29/31),the most frequent Chinese medicine pairing was "Rhizoma Coptis-Akinomia chinensis"(24/31),and the Chinese medicine pairing with Rhizoma Coptis-Akinomia chinensis was the most frequent,which was consistent with the record in the Chinese Pharmacopoeia(2020 edition).(2)Bioinformatics:(1)Xianglian pill has a certain effect on lipid metabolism regulation pathway.(2)FASN was highly expressed in the tumor tissues of 7 types of cancer.(3)The-log2(TPM+1)ratio of FASN expression in human colorectal adenocarcinoma tissues and normal intestinal tissues is 6.89:5.39(p < 0.001).(4)The survival period of high FASN expression group was significantly longer than that of low FASN group(p < 0.05).(5)FASN expression in colon cancer patients with normal body weight(BMI < 25)was higher than that in obese(BMI ≥ 25)colon cancer patients(p < 0.05).In obese colon cancer patients,the short-term survival rate of patients with high FASN expression is higher than that of patients with low FASN expression,but the long-term survival rate is inferior to that of patients with low FASN expression(p < 0.05).(3)Structural bioinformatics: the affinity between berberine and FASN has 5 active pocket locations.2.Animal experiments(1)Body weight and tumor weight: Compared with the blank control group,the body weight of nude mice in the in-situ tumor group was reduced;Compared with the in situ tumor group,the body weight of the PA group increased.Compared with PA group,the body weight of nude mice in each drug group was reduced,and the effect of high dose berberine on reducing fat was the most obvious.Compared with PA group,Xianglian pill,berberine and orlistat all reduced tumor mass.Among them,the positive drug orlistat has the best anti-tumor effect.(2)Blood lipid: Total cholesterol in nude mice was significantly down-regulated after inoculation of colon tumor in situ,with statistical significance(p < 0.05).Compared with the in-situ tumor group,Palmitic acid can up-regulate the serum Triglyceride(TG),Total cholesterol(TC),Low density lipoprotein-cholesterol(LDL-C)in nude mice with colon cancer,the difference was statistically significant(p < 0.01).Compared with PA group,Xianglian pill could inhibit the increase of LDL-C induced by palmitic acid,and the difference was statistically significant(p < 0.01).The TC and LDL-C were decreased by medium dose berberine(p < 0.01,p < 0.05).High dose berberine could decrease TG and TC,and the difference was statistically significant(p < 0.01,p < 0.05).However,FASN inhibitor orlistat can only reduce LDL-C,and the difference is statistically significant(p< 0.05).(3)Intermediate products of palmitic acid generation: Compared with the in-situ tumor group,large amount of exogenous palmitic acid intake inhibited acetyl CoA,with statistical significance(p < 0.05).Compared with PA group,high dose berberine could increase the content of intermediate acetyl CoA,and the difference was statistically significant(p < 0.05).(4)FASN protein expression: The immunohistochemical experiment showed that,compared with the blank control group,FASN expression in the intestinal tissue of nude mice after inoculation with in situ colon cancer increased,with statistical significance(p< 0.05).Compared with the in-situ tumor group,FASN protein expression in intestinal tissue and tumor tissue was decreased after exogenous PA intake,ACC protein expression in normal intestinal tissue was increased,and ACC protein expression in tumor tissue was decreased,with statistical significance(p < 0.01,p < 0.001,p < 0.001,p < 0.001).After medication intervention,on the premise of exogenous palmitic acid dietary intake,low,medium and high doses of berberine and orlistat can all reduce FASN protein expression in normal intestinal tissues,with statistical significance(p < 0.01,p < 0.01,p < 0.05,p <0.001).Both low-dose berberine and orlistat could decrease the expression of ACC protein in intestinal tissue,with statistical significance(p < 0.05,p < 0.05).Both lowdose berberine and orlistat can reduce FASN protein expression in tumor tissues,with statistical significance(p < 0.01,p < 0.05).All Xianglian pills,low,medium and high doses of berberine and orlistat can reduce the expression of ACC protein in tumor tissues,and the differences were statistically significant(p < 0.01,p < 0.05,p < 0.001,p < 0.001,p < 0.001).Compared with Xianglian pill group,ACC protein of berberine(middle)group was upregulated in tumor tissue,with statistically significant difference(p < 0.01);FASN protein of intestinal tissue was down-regulated,with statistically significant difference(p < 0.01).3.Cell experiments(1)IC50 value: With the increase of berberine concentration(0-200 μM),the inhibition rate(IR)of SW480 cells increased significantly.In the same intervention period,compared with 0 μM,the OD values of berberine at all concentrations decreased,and the differences were statistically significant(p < 0.01).After 48 h of intervention,the IC50 value of berberine was 49.13 μM,and the concentration of berberine in subsequent experiments was 50 μM.(2)FASN protein expression: compared with the cell control group,the protein expression of FASN was significantly reduced in the 32 μM PA group,with statistical significance(p < 0.01).Compared with the 32 μM PA group,the expression of FASN in the 50 μM berberine +32 μM palmitic acid group was significantly decreased,with statistical significance(p < 0.01).Compared with the control group,low,medium and high doses of berberine can significantly down-regulate the expression of FASN protein level in SW480 cells(p < 0.001).Compared with the cell control group,low dose palmitic acid significantly down-regulated the expression of FASN protein level in SW480 cells,with statistical significance(p < 0.001).Compared with the low dose PA group,low,medium and high berberine and orlistat combined with low dose PA can significantly reduce the expression of FASN protein level,with statistical significance(p < 0.001,p < 0.05).Conclusion1.Literature mining and bioinformatics:(1)The formulation of Xianglian Pill has inheritance and continuity,which can regulate the lipid metabolism pathway.(2)The expression of FASN in tumor tissues is significantly higher than that in normal tissues,FASN is related to the occurrence of colorectal adenocarcinoma tumors,and is related to the survival time and prognosis of patients with colorectal adenocarcinoma.(3)Berberine,the active ingredient of Xianglian pill,has a binding site with FASN.2.Animal experiments:(1)Xianglian pill,berberine,orlistat(FASN inhibitor)and high concentration palmitic acid diet all inhibited orthotopic transplanted colon cancer tumors in nude mice.(2)Berberine can significantly down-regulate the expressions of FASN and ACC proteins in tumor tissues,and inhibit the increase of acetyl CoA which is the intermediate product caused by exogenous PA intake,while Xianglian pill has no significant effect.3.Cell experiment:(1)Berberine inhibited the proliferation of SW480 cells with IC50 value of about 50 μM in a time and dose dependent manner.(2)The specific mechanism by which berberine inhibits colon cancer cell proliferation through lipid lowering is related to its down-regulation of FASN protein expression.