[Molecular mechanism of total flavonoids in Isodon amethystoides on adjuvant arthritis in rats].

Our preliminary study showed that the total flavonoids in Isodon amethystoides(TFIA), a local medicinal herb in Suzhou, had a certain therapeutic effect on adjuvant arthritis, and this therapeutic effect may be achieved through the up-regulation of miR-152 expression. In this paper, the molecular mechanism of TFIA on the pathogenesis of adjuvant arthritis(AA) rats was further studied. AA rats were prepared with complete Freund's adjuvant, and then treated with TFIA by intragastric administration. Real-time qPCR was used to detect the effects of TFIA on the negative regulatory loop of miR-152, methylase DNMT1 and methyl-CpG binding protein MeCP2 in fibroblast like synoviocytes(FLS) of AA rats, as well as the effects of TFIA on the classic Wnt signaling pathway and the expression of fibronectin gene in AA rats. Intragastric administration of TFIA significantly inhibited the expression of DNMT1 and reversed the negative regulatory loop composed of miR-152, DNMT1 and MeCP2 in the pathology of AA rats. After transfection of miR-152 inhibitors into the FLS in treatment group, DNMT1 expression was significantly restored. TFIA significantly up-regulated the expression of SFRP4 and inhibited the expression of ß-catenin, C-myc and ccnd1, the key genes of canonical Wnt signaling pathway. TFIA also significantly inhibited the expression of fibronectin, an AA gene. The effect of TFIA on the expression of SFRP4, ß-catenin, C-myc, ccnd1 and fibronectin was reversed after transfection with miR-152 inhibitors in the treatment group FLS. TFIA may inhibit the DNMT1 expression, up-regulate the SFRP4 expression, inhibit the expression of classical Wnt signaling genes ß-catenin, C-myc, and ccnd1 as well as the RA gene fibronectin expression through the up-regulation of miR-152 expression.

[Simultaneous Determination of Four Bioactive Components in Chrysanthemi Flos by Quantitative Analysis of Multi-Components by Single Marker].

Objective: To establish a method of simultaneous determination of chlorogenic acid,galuteolin,quercetin and acacetin in Chrysanthemi Flos by quantitative analysis of multi-components by single marker( QAMS),and determine the application value of QAMS in the quality control of Chrysanthemi Flos. Methods: The analysis was established to use the Shim-pack C18( 250 mm × 4. 6 mm,5 µm),the mobile phase of water contained acetonitrile( A)-0. 1% phosphoric acid aqueous solution( B) in a gradient elution manner,the flow rate was 1. 0 m L / min,the column temperature was 30 ℃,the injection volume was 10 µL,the detection wavelength was set at348 nm. A characteristic spectrum was used for identification of four components. By using chlorogenic acid as reference,the relative corrective factors( RCF) of the other three components with reference to chlorogenic acid were calculated. The method was evaluated by comparison of the quantitative results through external standard method and QAMS method. Results: The RCF of galuteolin, quercetin and acacetin with reference to chlorogenic acid were 0. 6865,0. 9976 and 0. 6665,and the RSD( n = 6) were 0. 1439%,0. 2512%,0. 3971%,respectively. The results from QAMS method were not significantly different from those from external standard method. Conclusion: The method with a single marker is accurate and feasible to evaluate the quality of galuteolin,quercetin and acacetin as reference to chlorogenic acid in Chrysanthemi Flos by QAMS.

MicroRNA-663 activates the canonical Wnt signaling through the adenomatous polyposis coli suppression.

Rheumatoid arthritis (RA) is a symmetrical polyarticular autoimmune disease of unknown etiology. In this present study, we observed that the adenomatous polyposis coli (APC) expression is down-regulated and the expression of microRNA (miR)-663 increased significantly in synovium from RA patients compared with control. Target gene prediction for miR-663 revealed that the mRNA of APC gene, which is a member of the canonical Wnt signaling pathway, has a miR-663 binding site in its 3'-untranslated region (3'UTR). The result showed that increased miR-663 suppressed the APC expression significantly, and this down-regulation of APC expression triggered the activation of canonical Wnt signaling through accumulation of ß-catenin in fibroblast-like synoviocytes (FLS). In addition, increased miR-663 induced the FLS proliferation and the expression MMP3 and fibronectin during disease development. Therefore, miR-663 can be considered as a critical regulator of RA pathogenesis and can be utilized for developing miRNA-based therapeutic agents for RA patients.

DNMT1 activates the canonical Wnt signaling in rheumatoid arthritis model rats via a crucial functional crosstalk between miR-152 and the DNMT1, MeCP2.

In previous study, we identified that microRNA (miR)-152 expression was down-regulated in RA model rats, and overexpression of miR-152 inhibited the canonical Wnt signaling through the DNA methyltransferase (DNMT1) inhibition. However, the exact molecular mechanisms of DNMT1 were unclear. In this work, we investigate whether DNMT1 affects the pathogenesis of RA model rats and targets the miR-152 promoter. The effects of DNMT1 on the canonical Wnt signaling, the pathogenesis of RA model rats and the SFRP1 expression were detected by the real time qPCR, Western blotting, ELISA, MTT and viable cell number assay. The interaction between miR-152 and DNMT1, methyl CpG binding protein 2 (MeCP2) was investigated by real time qPCR and chromatin immunoprecipitation (ChIP). Our results revealed that increased DNMT1 activated the canonical Wnt signaling could not only by targeting SFRP4 may also by SFRP1 in RA model rats. Furthermore, treatment of DNMT1 inhibitor, 5-aza-2'-deoxycytidine (5-azadC), or knockdown of DNMT1, or knockdown of MeCP2 led to increased miR-152 expression by reversion of its promoter hypermethylation, DNMT1 and MeCP2 binding to the CpG islands of miR-152 promoter. Interestingly, it is proved a synergistic inhibition effect of DNMT1 and MeCP2 in this process. Moreover, overexpression of miR-152 could inhibit DNMT1 expression and result in a decrease of DNMT1 and MeCP2 binding to miR-152 promoter, and inhibition of miR-152 expression would reverse it. These observations demonstrate a crucial functional crosstalk between miR-152 and the DNMT1, MeCP2 by a double-negative circuit involved in the pathogenesis of RA model rats.

miR-375 regulates the canonical Wnt pathway through FZD8 silencing in arthritis synovial fibroblasts.

Whether the rheumatoid arthritis (RA) pathogenesis is regulated by microRNA (miRNA) is not entirely clear. In this study, we found that miR-375 was down-regulated significantly in fibroblast-like synoviocytes (FLS) in adjuvant-induced arthritis (AIA) rat model compared with control. Because the web-based software TargetScan and PicTar predict Frizzled 8 (FZD8) as the target of miR-375, we investigated whether up-regulated miR-375 plays a role in the activation of the canonical Wnt signaling by targeting the FZD8. Furthermore, the purpose of the present experiments was also to determine the role of miR-375 in the pathogenesis of AIA rat model and to ascertain the effects of FZD8 in this process. Real time qPCR, Western blotting, ELISA and ChIP assay were used to assess the inhibited role of miR-375 in the pathogenesis of AIA rat model and the canonical Wnt signaling. RNA interference was also used to detect the role of knockdown of dephosphorylated ß-catenin. Luciferase reporter gene and related methods were performed to determine the FZD8 as the target of miR-375. The increased miR-375 inhibited the pathogenesis of AIA rat model as indicated by decreases in the several disease markers, such as MMP3 and fibronectin. Interestingly, miR-375 also inhibited the canonical Wnt signaling, and the stabilized form of ß-catenin blocked the miR-375 effects. FZD8 was identified as the target of miR-375 in AIA rat model by the firefly luciferase reporter gene. In summary, our results demonstrate that miR-375 regulates the pathogenesis of AIA rat model through the canonical Wnt signaling pathway. This discovery may provide new targets for therapeutic intervention to benefit RA patients.

Giardia intestinalis: effects of Pulsatilla chinensis extracts on trophozoites.

Pulsatilla chinensis is a medicinal root plant that has been used to treat a wide range of disease conditions. Our study determined the antiprotozoal activity of various P. chinensis extracts and fractions against Giardia intestinalis including their effects on parasite growth, cell viability, adherence, and morphology. Ethyl acetate extracts (IC50 = 257.081 µg/ml) were the most active to inhibit the growth of G. intestinalis followed by aqueous extract (PWE), saponins, and n-butanol extract. The PWE and ethyl acetate extract inhibited G. intestinalis trophozoites adherence after 3 h of incubation and killed almost 50 % of the parasite population in a time-dependent manner. Changes in morphology, presence of precipitates in the cytoplasm, dissolved cytoplasm with large vacuole, break of flagella and ventral disk, membrane blebs, and intracellular and nuclear clearance of the treated trophozoites were observed by scanning and transmission electron microscopy. We demonstrated that P. chinensis induced these changes in G. intestinalis morphology and consequently has potential therapeutic use against giardiasis.

[Study on tissue culture of pasqueflower].

OBJECTIVE: To speed up seedling production of pasqueflower (Puzlsatilla chinenses) and their modernization in pasqueflower. METHOD: With tissue culture method, primary culture of different explants, culture of cluster buds and their rooting culture were conducted on medium of treatment combinations of adding different hormones. RESULT: The appropriate medium for different culture stages were MS + 6-BA 1.0-3.0 mg x L(-1) + NAA 0-0.05 mg x L(-1) + Sucrose 30 g x L(-1) in primary culture, MS + 6-BA 0.2 mg x L(-1) + NAA 0.02 mg x L(-1) + BR 0.00001 mg x L(-1) + Sucrose 30 g x L(-1) in differentiation and subculture of cluster buds, 1/2 MS + NAA 0.4 mg x L(-1) + Sucrose 20 g x L(-1) in rooting. CONCLUSION: Applying stem tip and flower buds as explants, high frequency propagation of seedlings can be achieved with plant tissue culture in Pasqueflower.