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ObjectiveTo induce the rat model of ulcerative colitis （UC） with spleen-kidney Yang deficiency and liver depression， and explore the efficacy and mechanism of Sishenwan combined with Tongxie Yaofang （SSW&TXYF） based on the therapeutic principles of tonifying spleen， soothing liver， warming kidney， and astringing intestine. MethodSixty male SD rats were randomized into normal， model， mesalazine， and high-， medium-， and low-dose SSW&TXYF groups. The rats in other groups except the normal group were administrated with Sennae Folium decoction and hydrocortisone and received tail clamping for 14 days. On day 14， rats received enema with TNBS-ethanol solution to induce UC. The rats were administrated with corresponding drugs from day 15 of modeling， and the body weight and mental state were observed and recorded. The sucrose preference test was performed from day 25. On day 28， the rectal temperature was measured， and the rats were administrated with 3% D-xylose solution at a dose of 10 mL·kg-1 by gavage. Blood was sampled 1 h later， from which the serum was collected for measurement of the D-xylose content. The serum， hippocampus， and colorectum samples of rats were collected on day 29. The levels of gastrin （GAS）， adrenocorticotropic hormone （ACTH）， corticosterone （CORT）， cyclic adenosine monophosphate （cAMP）， cyclic guanosine monophosphate （cGMP）， interleukin （IL）-4， tumor necrosis factor （TNF）-α， and interferon （IFN）-γ in the serum and 5-hydroxytryptamine （5-HT） in the hippocampus were determined by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining was employed to reveal the colonic lesions. The mRNA and protein levels of p38 mitogen-activated protein kinase （MAPK）， extracellular signal-regulated kinase （ERK）， and c-Jun N-terminal kinase （JNK） in the colon tissue were determined by Real-time PCR and Western blot， respectively. ResultCompared with the normal group， the model group showed decreased body weight， anal temperature， and D-xylose content in the serum and increased GAS content （P<0.01）. The modeling led to cAMP/cGMP unbalance and decreased the ACTH and CORT content in the serum （P<0.01）， the preference for sucrose water， and the 5-HT content in the hippocampus （P<0.01）. Moreover， it shortened the colorectal length and caused massive infiltration of inflammatory cells and severe structural damage in the colon tissue. High， medium， and low doses of SSW&TXYF improved above indicators （P<0.05， P<0.01）， reduced inflammatory infiltration， and repaired the pathological damage of the tissue. Compared with the normal group， the model group showed lowered IL-4 level （P<0.01） and elevated TNF-α and IFN-γ levels （P<0.05， P<0.01） in the serum， as well as up-regulated expression of p38 MAPK， ERK， and JNK （P<0.05， P<0.01）. Compared with the model group， SSW&TXYF elevated the IL-4 level （P<0.01）， lowered the TNF-α and IFN-γ levels （P<0.05， P<0.01）， and down-regulated the mRNA and protein levels of p38 MAPK， ERK， and JNK （P<0.05， P<0.01）. ConclusionA rat model of UC with spleen-kidney Yang deficiency and liver depression was successfully established. SSW&TXYF can significantly mitigate this syndrome by reducing the inflammatory response in the colon and inhibiting the MAPK pathway.
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ObjectiveTo verify the anti-oxidative stress effect of Huangqintang based on the nuclear factor E2-related factor 2 （Nrf2） signaling pathway by using Caco-2 cells as a carrier and RNA interference （RNAi） technology with in vitro experiments. MethodThe Caco-2 cells in the logarithmic growth phase were transfected with siRNA to construct siRNA Caco-2 cells. After normal Caco-2 cells and siRNA Caco-2 cells were incubated with Huangqintang of different doses， RNA and protein were extracted. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the mRNA and protein expression of heme oxygenase-1 （HO-1）， NAD（P）H quinone oxidoreductase 1 （NQO1）， glutathione S-transferase （GST）， Kelch-like ECH-associated protein 1 （Keap1）， and Nrf2. Meanwhile， the activities of superoxide dismutase （SOD） and GSH-Px， as well as the expression levels of malondialdehyde （MDA） and reactive oxygen species （ROS）， were detected by the colorimetric method and the probe method. ResultCompared with the results in the normal group， only the 400 mg·L-1 Huangqintang group and the sulforaphane （SFN） group could reduce the content of ROS and MDA in Caco-2 cells （P<0.01）， while the activities of SOD and GSH-Px in the cells of the Huangqintang groups and the SFN group showed an upward trend. Furthermore， there were significant differences in the 400 mg·L-1 Huangqintang group/the SFN group and the normal group （P<0.01）. Meanwhile， the protein and mRNA expression levels of HO-1， GST， Keap1， NQO1， and Nrf2 showed an upward trend in all groups （P<0.05， P<0.01）. After transfection， compared with the normal group， the model group showed increased content of MDA and ROS， blunted activities of GSH-Px and SOD， and reduced protein and mRNA expression of HO-1， GST， Keap1， and NQO1 （P<0.05， P<0.01）. After drug incubation， compared with the model group， the SFN group showed potentiated SOD activity， and the SFN group and the Huangqintang groups showed enhanced GSH-Px activity （P<0.01）. Moreover， the activities of SOD and GSH-Px in the 400 and 200 mg·L-1 Huangqintang groups and the SFN group showed an upward trend （P<0.01）， and the content of MDA in the 400 mg·L-1 Huangqintang group and the SFN group showed a downward trend. ROS decreased in all groups with drug intervention （P<0.01）， and the protein and mRNA expression of HO-1， GST， Keap1， NQO1， and Nrf2 increased to varying degrees （P<0.05， P<0.01）. ConclusionHuangqintang can play an anti-oxidative stress role by regulating the Nrf2 pathway.
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ObjectiveTo explore the anti-inflammatory mechanism of Huangqintang based on the inflammation model in RAW264.7 cells. MethodHuangqintang was prepared and the safe dose to RAW264.7 cells was screened out. The RAW264.7 cells were seeded in 24-well plates and incubated with Huangqintang and lipopolysaccharide （LPS）， successively. The concentrations of nitric oxide （NO）， interleukin （IL）-6， tumor necrosis factor （TNF）-α， and prostaglandin E2 （PGE2） were measured by Griess assay and enzyme-linked immunosorbent assay （ELISA）， respectively. Meanwhile， RAW264.7 cells were inoculated in 6-well plates， and normal group， LPS group， LPS+Huangqintang group， nuclear factor-κB （NF-κB） p65 inhibitor PDTC group， p38 mitogen-activated protein kinase （MAPK） inhibitor SB203580 group， extracellular signal-regulated kinase （ERK） inhibitor PD98059 group， c-Jun N-terminal kinase （JNK） inhibitor SP600125 group， and Janus kinase （JAK） inhibitor AG490 group were set up. After the cells were incubated with corresponding inhibitors and Huangqintang and stimulated by LPS， RNA and protein were extracted. The mRNA and protein expression levels of NF-κB p65， p38 MAPK， ERK， JNK， and JAK were detected by Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively， to explore the anti-inflammatory mechanism of Huangqintang by regulating the NF-κB， MAPK， and JAK/signal transducer and activator of transcription protein （STAT） signaling pathways. ResultAfter stimulation with LPS， the concentrations of NO， IL-6， TNF-α， and PGE2 in the cells of the model group increased significantly（P<0.05，P<0.01）. Compare with the model group， after incubation with Huangqintang， the secretion of NO， IL-6， TNF-α， and PGE2 showed a downward trend （P<0.05，P<0.01）. Compared with the normal group， the model group showed increased mRNA expression of p38 MAPK， ERK， JNK， JAK， and NF-κB p65 and total protein expression in cells after stimulation with LPS （P<0.05，P<0.01）. Compare with the model group，after incubation with Huangqintang， the total protein and mRNA expression of p38 MAPK， ERK， JNK， JAK， and NF-κB p65 in inflammatory cells decreased （P<0.05，P<0.01）. Meanwhile， the expression of NF-κB p65 total protein and mRNA in each inhibitor group showed a downward trend （P<0.05，P<0.01）. ConclusionHuangqintang can inhibit the inflammatory response through the NF-κB， MAPK， and JAK-STAT signaling pathways.
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ObjectiveTo evaluate the pharmacodynamic effect of Huangqintang （HQT） on ulcerative colitis （UC） model mice and investigate its protective effect against UC by regulating intestinal flora. MethodMale Balb/c mice were randomly divided into control group，model group， high-， medium-， and low-dose HQT groups （20， 10， 5 g·kg-1）， flora interference group， flora interference model group， and flora interference-drug treatment group （HQT， 20 g·kg-1）. The flora interference model was constructed through intragastric administration of antibiotics （200 mg·kg-1 bacitracin and 200 mg·kg-1 vancomycin） for 8 d， and the UC model was constructed by allowing mice with free access to 3% dextran sulfate sodium （DSS） solution for 7 d. HQT was administered for 7 d. After the experiments， the mice were sacrificed， and blood， colon， and feces were collected. Hematoxylin-eosin （HE） staining was performed to observe the colonic lesions. The serum levels of interleukin （IL）-4， IL-6， IL-10， and tumor necrosis factor （TNF）-α were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression of Claudin1， MUC1， Occludin， and zonula occludens-1（ZO-1） in colon tissues was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. The fecal DNA of mice was extracted and analyzed by high-throughput sequencing. ResultCompared with the normal group， the model group showed increased serum content of IL-4， IL-6， and TNF-α （P<0.05， P<0.01） and decreased IL-10 （P<0.05）. Compared with the model group， the HQT groups displayed decreased serum levels of IL-4， IL-6， and TNF-α （P<0.05， P<0.01）， increased IL-10 content （P<0.01）， increased mRNA and protein expression levels of Claudin1， MUC1， Occludin， and ZO-1 （P<0.05， P<0.01）. After flora interference， the diversity and abundance of intestinal bacteria decreased. To be specific， Proteobacteria increased （P<0.01）， and Firmicutes and Bacteroidetes decreased （P<0.01）. After UC induction by DSS， Bacteroidetes and Tenericutes decreased （P<0.05）. The high-， medium-， and low-dose HQT groups showed increased Bacteroidetes and Tenericutes （P<0.05， P<0.01） and decreased Firmicutes （P<0.05）. Additionally， the abundance of Lactobacillus， Lachnospiraceae NK4A136 group， Escherichia-Shigella， and Helicobacteris was positively proportional to the dose of HQT. ConclusionHQT can inhibit the inflammatory response of UC mice， restore the imbalance of intestinal flora， and repair the damaged intestinal mucosal barrier.
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Ulcerative colitis （UC） is a chronic intestinal disease with unknown etiology， with main symptoms of abdominal pain， diarrhea， mucus， pus， and blood in the stool. It can be accompanied by various complications and has a high risk of developing to colon cancer. In recent years， the incidence of UC and related colon cancer has been increasing， which seriously affects human health and quality of life. The operation， immunosuppressant， etc. are the main approaches in the modern clinical treatment of UC and related colon cancer， but these methods all have different toxic and side effects， and the therapeutic effect is not ideal. For many years， traditional Chinese medicine （TCM） has attracted much attention in the treatment of UC and related colon cancer due to its slightly toxic side effects and remarkable curative efficacy. Huangqintang， derived from the Shang Han Lun （伤寒论）， is composed of Scutellariae Radix， Paeoniae Radix Alba， Glycyrrhizae Radix et Rhizoma， and Jujubae Fructus with the functions of clearing heat， checking diarrhea， harmonizing the middle， and relieving pain， and has a significant effect on the treatment of UC. Huangqintang has complex compositions and plays roles with multiple targets and pathways. According to the literature and the research results of this research group for many years， it was found that the mechanism of Huangqintang in the treatment of UC and related colon cancer was presumably related to the protection of the intestinal mucosal barrier， inhibition of inflammatory response， promotion of mitophagy， inhibition of oxidative stress， regulation of intestinal flora， cell cycle， and gene expression， suppression of cell proliferation， and promotion of apoptosis. To provide theoretical references for an in-depth study of the mechanism and clinical use of Huangqintang， this paper reviewed the research advances in recent years.
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ObjectiveTo investigate the efficacy of Huangqintang on mouse models of colitis-associated colon cancer （CAC） and explore the mechanism of Huangqintang in regulating immune function and inflammatory response， inhibiting abnormal cell proliferation， and delaying or inhibiting CAC formation in CAC. MethodC57BL/6J mice were randomly divided into a normal group， model group， mesalazine group， and high- and low-dose Huangqintang groups according to body weight， with 12 mice in each group. Except for the normal group， the rest of the mice were given two intraperitoneal injections of 10 mg·kg-1 azomethane （AOM） and allowed to drink 1.5% dextran sodium sulfate （DSS） freely for seven days and water normally for two weeks. Then， two cycles of ''DSS-drinking water'' were repeated. During the administration of DSS， mice in the normal group and model group were given gavage in equal doses of pure water. Mice in the mesalazine group were given 150 mg·kg-1·d-1 mesalamine suspension for gavage， and mice in the high- and low-dose Huangqintang groups were given 18 and 9 g·kg-1·d-1 Huangqintang for gavage， respectively. Each group was given one dose daily until the end of three cycles. After the intervention， the body weight， colon length， and number of colon tumors in each group were measured， and disease activity index （DAI） scores were performed. The serum contents of interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-4 （IL-4）， interleukin-10 （IL-10）， and gastrointestinal tumor marker carbohydrate antigen-199 （CA199） were detected by enzyme linked immunosorbent assay （ELISA）. The colonic lesions were observed by hematoxylin-eosin （HE） staining. The expression of proliferative cell-associated antigen （Ki67） was observed by immunohistochemistry. The expression of T lymphocyte subsets （CD3+， CD4+， CD8+， and CD49b+） in mouse plasma was detected by flow cytometry. Fluorescein isothiocyanate-D （FITC-D） content in mouse serum was detected by fluorescent labeling method. The Western blot method was used to detect the expression of Cyclin D1， cyclin-dependent kinase 2 （CDK2）， cyclin-dependent kinase 4 （CDK4）， and tightly junction-related Occludin and Claudin-1. ResultCompared with the normal group， the body weight of mice in the model group decreased. DAI score increased significantly， and the colon became shorter. Pro-inflammatory factors such as IL-6， TNF-α， and IL-1β increased， and IL-6 and TNF-α were significantly increased （P<0.05）. The inflammatory factor IL-4 （P<0.05） and IL-10 were significantly reduced， and the tumor marker CA199 was significantly increased （P<0.01）. HE staining showed that colon lesions， intestinal mucosal epithelial defects with a large number of inflammatory infiltrates， serious crypt structure damage， and glandular arrangement disorder were observed in the model group. Ki67 positive granules were expressed in large areas of colonic tissue. The serum CD4+ and CD4+/CD8+ of mice in the model group decreased significantly （P<0.05）， and CD8+ increased significantly （P<0.05）. The plasma content of FITC-D in the model group was significantly increased （P<0.05）， and the expression of Cyclin D1， CDK2， and CDK4 proteins in colon tissue was significantly increased （P<0.05， P<0.01）. In addition， the expression of Occludin and Claudin-1 was significantly decreased. Compared with the model group， the body weight of mice in the mesalazine group and the high- and low-dose Huangqintang groups increased. DAI score decreased， and the colon became longer. IL-6， TNF-α， and IL-1β expression decreased （P<0.05， P<0.01）， but there was no significant change in IL-4 and IL-10. The content of CA199 was significantly reduced （P<0.05）， and the colomatoid lesions and inflammatory infiltrates were reduced in the mesalazine group and the Huangqintang group. The crypt structure damage was lighter， and the positive expression of Ki67 was reduced. CD4+， CD4+/CD8+， and CD49b+ increased， and the difference was not statistically significant. FITC-D content decreased （P<0.05）. The expression of Cyclin D1， CDK2， and CDK4 decreased （P<0.05， P<0.01）， and Claudin-1 and Occludin protein expression increased in the high-dose Huangqintang group （P<0.05）. ConclusionHuangqintang has a certain delay and inhibitory effect on AOM/DSS-induced inflammatory cancer transformation， and its mechanism of action may be related to regulating immune function and inflammatory response， inhibiting the release of pro-inflammatory factors， repairing damaged intestinal barriers， inhibiting abnormal proliferation of colon cells， and intervening in the formation and development of CAC colon tumors.
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ObjectiveBased on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MSE） technique， we identified qualitatively the metabolites of aristolochic acid（AAs） in rat in order to analyze the metabolic differences between water extract of Aristolochiae fructus（AFE） and Aristolochic acid Ⅰ（AAⅠ）. MethodSD rats were selected and administered AFE（110 g·kg-1·d-1） or AAⅠ（5 mg·kg-1·d-1） by oral for 5 days， respectively. Serum， urine and feces were collected after administration. Through sample pretreatment， ACQUITY UPLC BEH C18 column（2.1 mm×100 mm， 1.7 μm） was used with the mobile phase of 0.01% formic acid methanol（A）-0.01% formic acid water（B， containing 5 mmol·L-1 ammonium acetate） for gradient elution（0-1 min， 10%B； 1-7 min， 10%-75%B； 7-7.2 min， 75%-95%B； 7.2-10.2 min， 95%B； 10.2-10.3 min， 95%-10%B； 10.3-12 min， 10%B） at a flow rate of 0.3 mL·min-1. Positive ion mode of electrospray ionization（ESI+） was performed in the scanning range of m/z 100-1 200. In combination with UNIFI 1.9.4.053 system， the Pathway-MSE was used to qualitatively analyze and identify the AAs prototype and related metabolites in biological samples（serum， urine and feces）， and to compare the similarities and differences of metabolites in rats in the subacute toxicity test between AFE group and AAⅠ group. ResultCompared with AAⅠ group， 6， 10， 13 common metabolites and 14， 20， 30 unique metabolites were identified in biological samples（serum， urine and feces） of AFE group， respectively. Moreover， the main AAs components always followed the metabolic processes of demethylation， nitrate reduction and conjugation. Compared with common metabolites in AAⅠ group， prototype components of AAⅠ in serum and most metabolic derivatives of AAⅠ［AAⅠa， aristolochic lactam Ⅰ（ALⅠ）a， 7-OHALⅠ and its conjugated derivatives］ in biological samples were significantly increased in AFE group（P<0.05， P<0.01）， except that the metabolic amount of ALⅠ in feces of AFE group was remarkably lowed than that of AAⅠ group（P<0.01）. In addition， a variety of special ALⅠ efflux derivatives were also identified in the urine and feces of the AFE group. ConclusionAlthough major AAs components in AFE all show similar metabolic rules as AAⅠ components in vivo， the coexistence of multiple AAs components in Aristolochiae Fructus may affect the metabolism of AAⅠ， and achieve the attenuating effect by increasing the metabolic effection of AAⅠ and ALⅠ.
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The quality control processes for herbal medicines have been problematic. Flavonoids are the major active components of Huangqin Tang (HQT, a traditional Chinese medicine formula). In this study, we used a combinative method approach consisting of chromatographic fingerprinting (high performance liquid chromatography; HPLC), quantitative methods and a pharmacodynamic evaluation model to analyze the flavonoids of HQT obtained from different sources. Ten batches of HQT were analyzed by the HPLC fingerprinting method and 26 common peaks were detected, of which 23 peaks corresponded with the chemical profile of HQT. In addition, 11 major compounds were identified by LC-MS analysis (liquid chromatography-tandem mass spectrometer; LC-MS (n) ) and quantified by the HPLC quantitative method approach. The studied 10 batches of HQT were found to be homogeneous in their composition with a similarity between 0.990 and 1.000. The distribution of the 11 identified compounds was found to be very similar among the batches. Only slight pharmacodynamic differences were detected between the different batches, confirming the homogeneity of HQT. The results of this study prove that the combination of chromatographic fingerprinting and quantitative analysis can be readily used for comprehensive quality control of herbal medicines.
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A simple and selective HPLC method for simultaneous determination and quantification of anthraquinones, lignans and flavonoids in Xiao-Cheng-Qi Tang (XCQT), Hou-Po-San-Wu Tang (HPSWT) and Hou-Po-Da-Huang Tang (HPDHT) was developed and validated. An Agilent Zorbax SB-C 18 (4.6 mm x 250 mm, 5 µm) column with the mobile phase of acetonitrile and 0.5% acetic acid aqueous solution in gradient elution mode was used. The flow rate was 1.0 mL · min(-1) at 30 °C, and injection volume was 10 µL. The detection wavelength was set at 254 nm and 294 nm simultaneously for the quantitative analysis. The current HPLC assay was validated for linearity, intra-day and inter-day precisions, accuracy, recovery and stability. The method was applied to the content comparison of the gallic acid, cinnamic acid, sennoside A, sennoside B, rhein, emodin, aloe-emodin, chrysophanol, physcion, magnolol, honokiol, narirutin, naringin, hesperidin, neohesperidin, hesperetin, naringenin and nobiletin in XCQT, HPSWT and HPDHT. The good linear equations of eighteen constituents were obtained within the investigated ranges (r > 0.998). The recovery of the method was 94.28%-99.89% and the precision was less than 5%. The sample was stable within 16 h. There were some differences between the contents of anthraquinones, lignans and flavonoids in analogous formulae about XCQT. XCQT contained the greatest abundance of anthraquinones and flavonoid, HPSWT contained the greatest abundance lignans. In conclusion, the methods are simple, low-cost, precise, accurate and reliable for the determination of eighteen constituents in analogous formulae about XCQT, and these results provide methodological support for its quality control.