Inhibitory Effect and Mechanism of Sishenwan-containing Serum on Aerobic Glycolysis in Human Colon Cancer Cells / 中国实验方剂学杂志

ObjectiveTo explore the effect and mechanism of Sishenwan-containing serum on aerobic glycolysis in human colon cancer HCT116 cells. MethodCell counting kit-8 （CCK-8） was used to detect the cell viability of colon cancer HCT116 cells after treatment with Sishenwan-containing serum （2.5%， 5%， and 10%） for 24， 48， 72 h. The concentration of lactic acid， the content of intracellular glucose， and the activity of hexokinase （HK） and fructose-6-phosphate kinase （PFK） in the cell culture medium were detected by the micro-method. The content of glucose transporter 1 （GluT1） mRNA was detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression of GluT1 and methyltransferase-like 3 （MettL3） was detected by Western blot. The expression of GluT1 in cells was detected by immunofluorescence and the level of N6-methyladenosine （m6A） RNA methylation was detected by colorimetry. ResultCompared with the normal serum， 2.5%， 5%， and 10% Sishenwan-containing serum had no significant effect on the viability of HCT116 cells at 24 h， while 10% Sishenwan-containing serum showed a significant inhibitory effect on the viability of HCT116 cells at 48 h （P<0.05）. Hence， 10% Sishenwan-containing serum was used in subsequent experiments， and the intervention time was 48 h. Compared with the normal serum， 10% Sishenwan-containing serum could reduce lactate production （P<0.05）， down-regulate glucose uptake （P<0.05）， and blunt the activities of HK and PFK， the key rate-limiting enzymes of glycolysis （P<0.05）. Meanwhile， 10% Sishenwan-containing serum could decrease the expression of GluT1 protein （P<0.01） and mRNA （P<0.05） and reduce the proportion of cells expressing GluT1 （P<0.01）. Compared with the normal serum， Sishenwan-containing serum also decreased the protein content of MettL3 （P<0.05） and the methylation level of m6A RNA （P<0.01）. ConclusionSishenwan can inhibit glycolysis in colon cancer cells， and its inhibitory mechanism may be related to reducing MettL3 overexpression， inhibiting m6A RNA methylation， and down-regulating GluT1 and the activities of intracellular aerobic glycolysis-related enzymes such as HK and PFK.

Effect of Sishenwan， Baitouweng Tang， and Lianlitang on Ulcerative Colitis in Rats / 中国实验方剂学杂志

Objective:To explore the mechanism of Sishenwan， Baitouweng Tang， and Lianlitang in the treatment of ulcerative colitis （UC）， and compare their efficacies on UC in rats. Method:Ninety SD rats of SPF grade were randomly divided into blank group （distilled water， 2 mL·d^-1） and experimental group. The rats in the experimental groups were administered with 2，4，6-trinitrobenzene sulfonic acid （TNBS） by clysis to induce the UC model. Subsequently， the model rats were divided into a model group （distilled water， 2 mL·d^-1）， positive group ［sulfasalazine （SASP）， 0.4 g·kg^-1·d^-1］， Sishenwan group （1.76 g·kg^-1·d^-1）， a Baitouweng Tang group （1.40 g·kg^-1·d^-1）， and Lianlitang group （2.13 g·kg^-1·d^-1） according to the random number table. The rats in each group were dosed at 2 mL·d^-1 for 14 days. The pathological score for colonic mucosa was detected. Cytokines were detected by the cytokine chip. The enzyme-linked immunosorbent assay （ELISA） was used to detect the free triiodothyronine （FT₃）， glucagon-like peptide-1 （GLP-1）， and corticosterone （CORT） in plasma， and neurotensin （NT）， substance P （SP）， vasoactive intestinal peptide （VIP）， and somatostatin （SST） in colon tissues. Result:Compared with the normal group， the model group showed increased colon mass-length ratio and pathological score for colonic mucosa （<italic>P</italic><0.01）， infiltration of massive lymphocytes， disordered or absent intestinal villi， elevated levels of cytokine-induced neutrophil chemoattractant-1/2<italic>α</italic>/<italic>β</italic>/3 （CINC-1/2<italic>α</italic>/<italic>β</italic>/3）， interleukin-1<italic>α</italic> （IL-1<italic>α</italic>）， interferon-inducible protein-10 （IP-10） and other factors in colon tissues （<italic>P</italic><0.05）， dwindled CORT and GLP-1 levels in plasma （<italic>P</italic><0.05）， and increased SP content in colon tissues （<italic>P</italic><0.05）. Compared with the results in the model group， the mucosal injury in the colon of rats in each drug group was relieved. The levels of IL-1<italic>α</italic>， IP-10， lipopolysaccharide-inducible CXC chemokine （LIX）， and L-selectin of rats in the Lianlitang group and Sishenwan group were reduced （<italic>P</italic><0.05）， and the CINC-3 and IL-17 levels were diminished in the Baitouweng Tang group （<italic>P</italic><0.05）. The levels of CINC-1/3， IL-1<italic>α</italic>， and IP-10 were reduced in the SASP group （<italic>P</italic><0.05）. The plasma FT₃ was up-regulated in the Lianlitang group， and the plasma GLP-1 levels were elevated in the three Chinese medicine groups （<italic>P</italic><0.05）. The VIP content in colon tissues of the Sishenwan group and Baitouweng Tang group was down-regulated （<italic>P</italic><0.05）， and the SST content in colon tissues of the SASP group was significantly up-regulated （<italic>P</italic><0.01）. Conclusion:The intervention of Lianlitang and Sishenwan on UC was significant， and the underlying mechanism of action might be related to inflammation inhibition and immune balance by regulating the cytokine network. The efficacy of Lianlitang was predominant， followed by Sishenwan and Baitouweng Tang.

Regulatory Effect of Volatile Oil from Sishenwan on TLR/MyD88 Signaling Pathway in Mice with Chronic Ulcerative Colitis / 中国实验方剂学杂志

Objective:To explore the underlying mechanism of volatile oil from Sishenwan in treating chronic ulcerative colitis through the Toll-like receptor （TLR）/myeloid differentiation factor 88 （MyD88） signaling pathway. Method:The BALB/c mice were randomly divided into a normal group （normal）， a model group ［dextran sodium sulfate （DSS）］， a Sishenwan volatile oil group， an Ershen pill volatile oil group， a Wuweizi powder volatile oil group， and a mesalazine control group. The chronic ulcerative colitis model was induced by DSS in mice. Seven days after intragastric administration， the efficacy was evaluated based on the body weight， colon weight， colon weight index， colon length， and pathological damage score under colonoscopy. The levels of interleukin （IL）-4， IL-10， IL-17A， IL-21， and interferon-<italic>γ </italic>（IFN-<italic>γ</italic>） in the supernatant of colon tissues were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the expression levels of proteins related to the TLR/MyD88 signaling pathway in the colon mucosa of mice， including TLR2， MyD88， Ras-related C3 botulinum toxin substrate 1 （Rac1）， IL-1 receptor-associated kinase 4 （IRAK4）， IRAK1， tumor necrosis factor receptor （TNFR）-associated factor 6 （TRAF6）， transforming growth factor-<italic>β</italic>-activated kinase 1 binding protein 1 （TAB1）， TAB2， mitogen-activated protein kinase kinase 6 （MKK6）， p38 mitogen-activated protein kinase （p38 MAPK）， and cyclic adenosine monophosphate response element-binding protein （CREB）. Result:Compared with the normal group， the model group showed decreased colon length， increased colon weight， colon weight index， and pathological damage score under colonoscopy， decreased IL-10 level in the colon tissues， increased IL-4， IL-17A， IL-21， and IFN-<italic>γ</italic> levels （<italic>P<</italic>0.05， <italic>P<</italic>0.01）， and up-regulated protein expression of TLR2， MyD88， Rac1， IRAK4， IRAK1， TRAF6， TAB1， TAB2， MKK6， p38MAPK， and CREB （<italic>P<</italic>0.01）. Compared with the model group， the Sishenwan volatile oil group showed increased colon length， reduced colon weight， colon weight index， and pathological damage score under colonoscopy， elevated IL-10 level in the colon tissues， decreased IL-4， IL-17A， IL-21， and IFN-<italic>γ</italic> levels （<italic>P<</italic>0.05， <italic>P<</italic>0.01），and down-regulated protein expression of TLR2， MyD88， Rac1， IRAK4， IRAK1， TRAF6， TAB1， TAB2， MKK6， p38MAPK， and CREB （<italic>P<</italic>0.05， <italic>P<</italic>0.01）. Conclusion:The volatile oil from Sishenwan can effectively improve the inflammatory response of chronic ulcerative colitis， which may be achieved by regulating the TLR/MyD88 signaling pathway.

Effect of Sishenwan on PI3K/Akt/mTOR Signal Pathway in Colonic Tissue of Rats with Ulcerative Colitis Model of Spleen Kidney Yang Deficiency / 中国实验方剂学杂志

Objective:To discuss the effect of Sishenwan on phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin（PI3K/Akt/mTOR） signaling pathway related genes and proteins in colon tissue and interleukin-1<italic>β</italic>（IL-1<italic>β</italic>） and interleukin-10（IL-10） expression levels in serum of ulcerative colitis （UC） model rats with spleen kidney Yang deficiency. Method:The 120 SPF Wistar rats were randomly divided into normal group and model group after 7 days of adaptive feeding in SPF laboratory. The model group were given dinitrobenzene sulfonate （DNBS）/ethanol solution enema+hydrocortisone subcutaneously injection+senna leaf gavage to establish UC model of spleen kidney yang deficiency. The rats who successfully established the model were randomly divided into five groups：model group， mesalazine （0.36 g·kg^-1） group， and Sishenwan high， medium and low dose （3.2，1.6，0.8 g·kg^-1） groups， the volume of which was 10 mL·kg^-1. The model group and the blank group were given distilled water of the same volume. Once a day for 21 days. Observe the general conditions of the rats daily， and record the weight， fecal traits and occult blood of the mice for disease activity index （DAI） scoring.Take the rat colon tissue to observe the gross morphology and colon injury， and score the colon mucosal injury index （CMDI）. Hematoxylin-eosin （HE） staining was used to observe pathological changes. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）was used to detect the expression level of PI3K， Akt， mTOR mRNA in colon tissue. The levels of IL-1<italic>β</italic> and IL-10 in serum were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the expression level of PI3K， phosphorylation （p）-PI3K， Akt， p-Akt， mTOR， p-mTOR protein in colon tissue. Result:Compared with blank group， the general survival status of the rats in model group was relatively poor， the DAI score and the CMDI index were significantly increased （<italic>P<</italic>0.01）. The intestinal mucosa partially disappears， the glands disappear， and a large number of inflammatory cells infiltrate and gather in the mucosal layer and the base layer in the pathological sections of the model group. The expression levels of PI3K， Akt， and mTOR mRNA were significantly increased （<italic>P</italic><0.01）. The IL-1<italic>β</italic> content was significantly increased and the IL-10 content was significantly decreased （<italic>P</italic><0.01）. The expression levels of p-PI3K，p-Akt and p-mTOR protein were significantly increased （<italic>P</italic><0.01）. Compared with the model group， the DAI scores of Sishenwan high and medium dose groups and mesalazine group decreased （<italic>P</italic><0.05）. The CMDI index of mesalazine group and the high， middle and low dose groups of Sishenwan was significantly reduced （<italic>P</italic><0.01）. Pathological sections of rats showed that the inflammatory cells in the drug group decreased， and the mucosal layer structure returned to normal to varying degrees. The mesalazine group and the Sishenwan medium-dose group had the best effects， and the mucosal structure was close to the blank control group. The expression levels of PI3K， Akt， mTOR mRNA in the high and medium dose groups of Sishenwan， mesalazine group and Akt mRNA in low dose group of Sishenwan were significantly reduced （<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression levels of PI3K and mTOR mRNA in low-dose group of Sishenwan decreased， but the difference was not statistically significant. The IL-1<italic>β</italic> content was significantly reduced and the IL-10 content was significantly increased in high， medium dose groups of Sishenwan and mesalazine groups （<italic>P</italic><0.05，<italic>P</italic><0.01）. The level of IL-1<italic>β</italic> decreased and the level of IL-10 increased in the low-dose group of Sishenwan， but the difference was not statistically significant. The expression levels of p-PI3K， p-Akt and p-mTOR protein in the high， medium， and low dose groups of Sishenwan and mesalazine group decreased to varying degrees， and the differences were statistically significant（<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Sishenwan has the effect of improving the general condition and intestinal mucosal damage of ulcerative colitis model rats with spleen and kidney Yang deficiency. The mechanism may be related to the inhibition of PI3K/Akt/mTOR signaling pathway.

Effect of Sishenwan on Toll-like Receptor 4 and IRAK-M Expression in Colonic Tissue of Rats with Ulcerative Colitis of Spleen-kidney Yang Deficiency Type / 中国实验方剂学杂志

Objective:To observe the Toll-like receptor 4(TLR4)and its negative regulating factorInterleukin-1 receptor-associated kinase-M(IRAK-M)in colonic mucosa of rats with experimental ulcerative colitis(UC), and to discuss the mechanism of the Chinese medicine Sishenwan. Method:The 90 Wistar rats were randomly divide into six groups, blank group, model group, sulfasalazine group(0.36 g·kg-1), Sishenwan low, medium and high-dose group(2.5, 5, 10 g·kg-1), 15 cases in each group. A rat model of UC was prepared by using a solution of trinitrobenzenesulfonic acid/ethano.The histopathological changes of colon were observed by hematoxylin-eosin (HE) staining. The contents of serum free triiodothyroid acid (FT3), serum free thyroxine (FT4), immunoglobulin (Ig) E and interleukin (IL)-2 were determined by radioimmunoassay. The activity of superoxide dismutase (SOD) in rat serum was determined by xanthine oxidation method. The activity of malondialdehyde (MDA) in serum of rats was determined by thiobarbituric acid (TBA) colorimetry. Result:Compared with blank group, intestinal mucosal injury score of rats in model group was significantly increased (PP3, FT4, IL-2 and SOD were significantly decreased (PPPPPP3, FT4, IL-2, and SOD contents were significantly increased (PPPPPConclusion:The unbalanced expressions of TLR4 and its negative regulating factor IRAK-M are connected with the pathogenesis of UC.Sishenwan can cure UC and control the expression of TLR4 and promote the expression of IRAK-M.

Mechanism of Sishenwan in Treatment of Ulcerative Colitis Based on Network Pharmacology and Bioinformatics / 中国实验方剂学杂志

Objective:To investigate the mechanism of Sishenwan in the treatment of Ulcerative Colitis and to explore the key targets and signal pathway on the treatment of Ulcerative Coltis based on network pharmacology and bioinformatics. Method:To obtain the active ingredients and predicted target genes of Sishenwan by searching the Encyclopedia of Traditional Chinese Medicine(ETCM) database, from the Gene Expression Omnibus (GEO) database and Database of Gene-disease Associations (DisGeNET), Online Mendelian Inheritance in Man (OMIM), Drug-targetDatabase (DrugBank), The Comparative Toxicogenomics Database (CTD) and Pharmacogenetics and Pharmacogenomics Knowledge Base (PharmGKB) disease database collection of Ulcerative Colitis target genes, the drug-disease protein target gene was obtained by intersecting the two and to display the results by Cytoscape software, through the algorithm of network topology to screen out the key target genes, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out on key target genes using Gene Ontology Enrichment Analysis Softword Toolkit (GOEAST) and The Database for Annotation, Visualization and Integrated Discovery (DAVID) online tools, analyze the mechanism of Sishenwan in preventing and treating Ulcerative Colitis combined with relevant literature. Result:There are 182 active ingredients and 611 predicted targets in Sishen Wans, 914 known disease targets related to the occurrence and development of Ulcerative Colitis were retrieved through the disease database, it was concluded that the effect of Sishenwan on Ulcerative Colitis mainly involves steroid hormone-mediated signal pathway, exogenous metabolic process, positive regulation of transcription of RNA polymerase II promoter, lipid metabolic process, detoxification of cell oxidant, signal transduction and other biological functions, and participates in arachidonic acid, drug metabolism-cytochrome P450, cytochrome P450 for xenobiotics, chemical carcinogenesis and other key signal pathways. Conclusion:Sishenwan has the characteristics of multi-target, multi-channel and multi-level in the treatment of Ulcerative Colitis. Its multiple signal pathways are directly or indirectly related, participating in lipid protein metabolism, drug metabolism and anti-cancer mechanism, etc. It exerts its efficacy through comprehensive intervention on the digestive system, circulatory system, immune system and other multiple systems of the body, thus being consistent with the comprehensive effect mechanism of Ulcerative Colitis induced by multiple factors.