Effect of total saponins from Panax japonicus on non-alcoholic steatohepatitis by regulating autophagy / 中国中药杂志

Non-alcoholic steatohepatitis(NASH) was induced by high-sugar and high-fat diet in mice to investigate the intervention effect of total saponins from Panax japonicus(TSPJ) and explore its possible mechanism. Mice were fed with high-sugar and high-fat diet to establish NASH model, and intervened with different doses of TSPJ(15, 45 mg·kg~(-1)). The animals were fed for 26 weeks. The histomorphology and pathological changes of liver tissues were observed by HE staining. The transcriptional expression levels of miR-199 a-5 p, autophagy related gene 5(ATG5) and inflammatory cytokines interleukin-6(IL-6), interleukin-1β(IL-1β) and tumor necrosis factor α(TNF-α) in mouse liver were measured by quantitative Real-time polymerase chain reaction(qRT-PCR). Western blot was used to detect the expression of autophagy-related proteins ATG5, P62/SQSTM1(P62), and microtubule-associated protein light chain 3(LC3)-I/Ⅱ proteins in mouse liver. The expression of P62 protein was detected by immunofluorescence staining. In order to verify the targeting regulation relationship between miR-199 a-5 p and ATG5, miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor were transfected into Hepa 1-6 cells, and the expression of ATG5 mRNA and protein was detected. pMIR-reportor ATG5-3'UTR luciferase reporter gene plasmid was constructed and co-transfected with miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor into Hepa 1-6 cells to detect luciferase activity. In vivo, HE staining in the model group showed typical fatty degeneration and inflammatory infiltration, with increased expression of miR-199 a-5 p and decreased expression of ATG5 mRNA and protein. The expression of autophagy-associated protein P62 increased significantly, the ratio of LC3Ⅱ/Ⅰ decreased, and the transcriptional expression of inflammatory factors increased significantly. After the intervention by TSPJ, the pathological performance of liver tissue was significantly improved, the expression of miR-199 a-5 p decreased and the expression of ATG5 mRNA and protein increased, the expression of autophagy-associated protein P62 decreased significantly, the ratio of LC3Ⅱ/Ⅰ increased, and the transcriptional expression of inflammatory cytokines IL-6, IL-1β and TNF-α decreased significantly. In vitro, it was found that the expression of ATG5 mRNA and protein and luciferase activity decreased significantly in miR-199 a-5 p overexpression cells, while after inhibition of miR-199 a-5 p expression, the expression level of ATG5 mRNA and protein and luciferase activity increased. The results showed that TSPJ can improve NASH in mice fed with high-sugar and high-fat diet, and its mechanism may be related to the regulation of miR-199 a-5 p/ATG5 signal pathway, the regulation of autophagy activity and the improvement of inflammatory response of NASH.

Therapeutic effect of total triterpenoids of Chaenomeles speciosa combined with omeprazole on gastric ulcer induced by indomethacin in rats / 中国中药杂志

The aim of this paper was to observe the combination therapy with total triterpenoids of Chaenomeles speciosa and omeprazole on indomethacin-induced gastric ulcer in rats, and explore its possible mechanism. Rats were randomly divided into normal group, model group, omeprazole monotherapy(3.6 mg·kg~(-1)) group, total triterpenoids of C. speciosa monotherapy(100 mg·kg~(-1)) group, total triterpenoids of C. speciosa and omeprazole combination therapy(100 mg·kg~(-1)+3.6 mg·kg~(-1)) group. Except for the normal group, the other groups were given indomethacin(20 mg·kg~(-1)) by oral once a day for 7 consecutive days. Then the treated groups were given corresponding drugs by gavage, once a day for 14 consecutive days. The next day after the last administration, half of the rats in each group were measured the gastric mucosal blood flow, gastric juice volume and serum TNF-α, IL-1β, IL-6, IL-4 and IL-10. After the remaining rats in each group were underwent pyloric ligation 4 hours after the last administration, the gastric endocrine volume, pH value and total acidity of gastric secretion were measured, then histological analysis was performed, MPO activity, cAMP content and histomorphological analysis were conducted. Real-time PCR was applied to detect the mRNA expressions of gastric tissue TNF-α,IL-1β, IL-6, IL-4, IL-10, VEGFA, A_(2A)R; the protein expressions of VEGFA, A_(2A)R, PKA, p-PKA, CREB, p-CREB, EGF, EGFR, p-EGFR, MUC6, TFF2 in gastric tissue were detected by Western blot. The results indicated that total triterpenoids of C. speciosa and omeprazole combination therapy might significantly increase gastric mucosal blood flow, gastric mucus volume, reduce gastric endocrine volume, secretion acidity and mucosal damage, decrease the levels of TNF-α,IL-1β and IL-6, increase the levels of IL-4 and IL-10 in blood and gastric tissue, inhibit the activity of MPO, increase the content of cAMP in gastric tissue, up-regulate the mRNA expressions of VEGFA, A_(2A)R and protein expressions of VEGFA, A_(2A)R, PKA, p-PKA, CREB, p-CREB, EGF, EGFR, p-EGFR, MUC6, TFF2 in gastric tissue, elevate p-PKA/PKA, p-CREB/CREB and p-EFGR/EFGR. Moreover, the combination therapy with total triterpenoids of C. speciosa and omeprazole was more obvious than those of two monotherapies. These aforementioned findings suggested that the combination therapy with total triterpenoids of C. speciosa and omeprazole on indomethacin-induced gastric ulcer have significant therapeutic effect on indomethacin induced gastric ulcer in rats, its mechanism might be related to regulating A_(2A)R/AKT/CREB, A_(2A)R/VEGFA, EGF/EGFR and MUC6/TFF2 signaling pathways, inhibiting pro-inflammatory factors, increasing gastric mucosal blood flow, up-regulating mucosal cell proliferation factors and promoting mucosal protective factors.

Effect of total triterpenoids of Chaenomeles speciosa on PPARγ/SIRT1/NF-κBp65 signaling pathway and intestinal mucosal barrier of ulcerative colitis induced by DSS in mice / 中国中药杂志

To observe the effect of total triterpenoids of Chaenomeles speciosa on PPARγ/SIRT1/NF-κBp65 signaling pathway and intestinal mucosal barrier of ulcerative colitis induced by dextran sulfate sodium (DSS) in mice, C57BL/6 mice were randomly divided into normal group, model group, total triterpenoids of C. speciosa (50, 100 mg·kg⁻¹) groups and sulfasalazine (250 mg·kg⁻¹) group. The ulcerative colitis (UC) model was induced by orally administering 2.5% DSS to the experimental mice, and the corresponding drugs were given to each group 3 days before the administration with 2.5% DSS. The normal group and the model group were given the equal volume of 0.5% carboxymethyl cellulose sodium solution by gavage continuously for 10 days, q.d. The general conditions of the mice were observed on a daily basis, and the disease activity index (DAI) score was recorded. On the 10th day after the treatment, mice were put to death, the contents of TNF-α, IL-1β, IL-6, IFN-γ, IL-4 and IL-10 in the blood were detected, colon length was measured, colon mucosa damage index (CMDI) score was calculated, and MPO activity detection and histomorphology analysis were conducted. Real-time PCR was applied to detect the mRNA expressions of E-cadherin, occluding,MUC2 and TFF3; the protein expressions of SIRT1, IKKβ, p-IKKβ, IκBα, p-IκBα and cytosol and nucleus PPARγ, NF-κBp65 in intestinal tissue were detected by western blot. The results indicated that total triterpenoids of C. speciosa (50, 100 mg·kg⁻¹) could significantly improve the general conditions of UC mice, reduce the DAI, CMDI and histopathological scores, increase the colon length, reduce the colonic mucosa ulcers, erosion and inflammatory infiltration, restore the normal intestinal mucosal barrier function, reduce the contents of TNF-α, IL-1β, IL-6, IFN-γ, increase the contents of IL-4 and IL-10 in the blood, inhibit MPO activity in colon tissue, up-regulate the mRNA expressions of E-cadherin, occludin, MUC2 and TFF3 in colon tissue, down-regulate the protein expressions of cytosol PPARγ, tissue p-IKKβ, p-IκBα and nucleus NF-κBp65 in the colon tissue, decrease the p-IKKβ/IKKβ and p-IκBα/IκBα ratios, up-regulate the protein expressions of nucleus PPARγ, tissue SIRT1 and cytosol NF-κBp65 (<0.05 or <0.01, respectively), with a dose-effect relationship between the total triterpenoids of C. speciosa treated groups. These findings suggested that total triterpenoids of C. speciosa had a significantly therapeutic effect on UC mice induced by DSS, its mechanism might be related to the regulation of PPARγ/SIRT1/NF-κBp65 signaling pathway, the inhibition of pro-inflammatory factor formation and the up-regulation of protein expression of protective factors.

Trametenolic acid B induces the programmed death of human gastric cancer HGC-27 cells / 中成药

AIM To study the programmed death effect of trametenolic acid B (TAB) against human gastric cancer HGC-27 cells.METHODS The proliferation of cells was examined by MTT assay;cell apoptosis rate and cell cycle were detected by flow cytometry;cell autophagy was observed by acridine orange staining and transmission electron microscope;expressions of autophagy-related proteins were detected by Western blot.RESULTS 10,20,40 μmol/L Trametenolic acid B had good growth-inhibitory effects on HGC-27 cells,blocked in G0/G1 phase,in a dose-dependent and time-dependent manner,but had no obvious effect on cell apoptosis.After treatment with 20 μmol/L trametenolic acid B for 24 h,a large number of autophagy vacuoles and autophagy bodies were observed under transmission electron microscope,and the proportion of cell autophagy was significantly increased with higher dose of TAB;TAB promoted the transformation of type Ⅰ microtubule-associated protein 1 light chain 3 (LC3 Ⅰ) to type Ⅱ microtubule-associated protein 1 light chain 3 (LC3 Ⅱ),up-regulated the expressions of myosin-like B-cell lymphoma-2 (Bcl-2) interacting protein (Beclin-1) and cyclin-dependent kinase inhibitor 1A (p21),and inhibited the CCND1 gene protein (CyclinD1) expression.CONCLUSION Trametenolic acid B can induce the autophagic death of HGC-27 cells,but it has no significant effect on cell apoptosis.

An analysis on the epidemic characteristics of tuberculosis drug resistance in Zhejiang Province / 预防医学

Objective To monitor the drug resistance of tuberculosis in Zhejiang Province and to provide scientific evidence for the control of drug resistant tuberculosis.Methods Thirty counties in Zhejiang Province were selected as sample during July 1,2013 and June 30 2014,and smear positive cases were selected to be monitored during study period. Results The rate of drug resistance was 30.88%,and the rate of multi drug resistance was 5.02%.The rate of the extensively drug -resistant tuberculosis was 0.32%.Among initial treatment cases,the rate of drug resistance was 29.22%,higher than 2008,and the rate of multi drug resistance was 3.21%.Among retreatment cases,the rate of drug resistance was 45.74%,and the rate of multi drug resistance was 21.28%.All kinds of monitored drugs were found resistance phenomenon.The drug resistant rate of SM was highest (15.28%),and aminoglycosides (2.35%)were relatively low.Conclusion The status of retreatment TB drug resistance suggested that we had reduced acquired TB drug resistance through implementation of DOTS strategy and standard short course chemotherapy treatment in Zhejiang Province. But it is still not optimistic to control TB drug resistance,and the status of initial treatment TB resistant suggested that resistant strains spread had not been effectively controlled in Zhejiang Province.So we should strengthen the early detection of drug resistant among TB patients,and to further improve the standard of conventional TB treatment.

Identification of Geranii herbae using ITS2 sequence / 中国药学杂志

OBJECTIVE: To provide the molecular basis for standard medicine market management, a new method to identify multi-origin traditional Chinese medicine Geranii herba was established by ITS2 sequence analysis. METHODS: As a prelude to species identification, Erodium stephaniahum willd., Geranium carolinianum L., Geranium wilfordii Maxim., together with their relatives adulterants, were sampled. A reference ITS2 sequence library was developed, including 82 ITS2 sequences of 28 species download from GenBank. Based on the intra-species, inter-species divergence, variable sites and the neighbor-joining (NJ) phylogenetic tree observed, a molecular identification method was established to the multi-origin herbs Geranium using ITS2 sequence. RESULTS: The NJ trees showed three origin species formed one monophyletic clade respectively; in addition any one of multi-origin herbs Geranium and its relatives adulterants could be distinguished clearly. Using this method, 18 random copies of Geranium herb collected from market were analyzed, 16 of which are genuine, 2 as adulterants. CONCLUSION: Therefore ITS2 sequence is suitable to be as a barcode to identify multi-origin herbs Geranium and their relatives adulterants.

Effect of mechanical stimulation on the expression of E-cadherin/cateninin complex in salivary adenoid cystic carcinoma / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of cyclic biaxial mechanical stimulation on adhesion of salivary adenoid cystic carcinoma(SACC) extracellular matrix (ECM) and expression of E-cadherin/cateninin complex in the SACC high metastasis cell lines ACC-M, SACC low metastasis cells line ACC-2, we observed the functions of mechanical stimulation in the adhesion of SACC cell-ECM and investigate the mechanism in the adhesion of ACC.</p><p><b>METHODS</b>Mechanical stimulation were applied to the cells for periods of 1, 3 and 6 hours every day, lasting for 2 days. The amplitude of mechanical stimulation applied to the cells were 1000, 4000 micro strain, at a frequency of 3 Hz. Unstrained cells were used as control. The expression of E-cadherin/cateninin complex on the cell of ACC-M, ACC-2 were studied with laser scanning confocal microscope and image analysis. SACC cell-ECM adhesion was assayed by MTT technique.</p><p><b>RESULTS</b>The results showed that expressions of alpha, beta, gamma-cateninin on the cell of ACC-2 were obviously higher than that ACC-M and E-cadherin on the cell of ACC-M were obviously higher than that ACC-2 without mechanical stimulation. Mechanical stimulation can change the expression of E-cadherin/cateninin complex on the cell of ACC-2 and ACC-M with time. The results also showed that cell-ECM adhesion on the cell of ACC-2 were obviously higher than that of ACC-M without mechanical stimulation. Mechanical stimulation can change the cell-ECM adhesion of ACC-2 and ACC-M with time.</p><p><b>CONCLUSION</b>Mechanical stimulation can change the adhesion of the SACC cell-ECM and expression of E-cadherin/cateninin complex of the SACC cell. We think it played an important role in metastasis of the cancer.</p>