Topotecan induces hepatocellular injury via ASCT2 mediated oxidative stress / El topotecán induce una lesión hepatocelular a través del estrés oxidativo mediado por el ASCT2

BACKGROUND: Topotecan is an anti-cancer chemotherapy drug with common side effects, including hepatotoxicity. In this study, we aim to investigate the mechanisms of topotecan-induced hepatocellular injury beyond conventional DNA damage. MATERIALS AND METHODS: Methyl Thiazolyl Tetrazolium (MTT) assay was used to detect the inhibitory effect of topotecan on cell proliferation. Western blot was used to detect protein expression. Flow cytometry assay was performed to determine apoptosis rate under topotecan treatment. ASCT2 overexpression was addressed using adenovirus vector. qRT-PCR and western blot assay were used to detect the expression of ASCT2. Glutamine uptake, intracellular glutathione (GSH) and reactive oxygen species (ROS) level were detected by glutamine detection kit, GSH detection kit and ROS detection kit respectively. RESULTS: MTT results showed that topotecan had an inhibitory effect on cell proliferation and induced apoptosis in both L02 and HepG2 cell lines. Topotecan inhibited the expression of glutamine transporter ASCT2 and the uptake of glutamine in both L02 and HepG2 cell lines. The uptake of glutamine and the GSH level was increased in both L02 and HepG2 cell lines after ASCT2 overexpression. The ROS level was inhibited by ASCT2 overexpression upon topotecan treatment in both L02 and HepG2 cell lines. Topotecan-induced hepatocellular apoptosis and proliferation inhibition were attenuated by ASCT2 overexpression in both L02 and HepG2 cell lines. CONCLUSION: Topotecan-induced hepatocytes death is dependent on ASCT2 down-regulation, which causes oxidative stress via inhibiting GSH production

ANTECEDENTES: El topotecán es un fármaco quimioterapéutico antineoplásico con efectos secundarios frecuentes, incluida la hepatotoxicidad. En este estudio nos proponemos investigar los mecanismos de la lesión hepatocelular inducida por el topotecán más allá del daño convencional del ADN. MATERIALES Y MÉTODOS: Se utilizó el ensayo de metil tiazolil tetrazolio (MTT) para detectar el efecto inhibitorio del topotecán sobre la proliferación celular. Se utilizó inmunoelectrotransferencia para detectar la expresión de las proteínas. Se realizó un ensayo de citometría de flujo para determinar la tasa de apoptosis con el tratamiento con topotecán. La sobreexpresión del ASCT2 se abordó utilizando un vector adenoviral. Se utilizaron la qRT-PCR y el ensayo de inmunoelectrotransferencia para detectar la expresión del ASCT2. La absorción de glutamina, el nivel de glutatión intracelular (GSH) y el nivel de especies reactivas del oxígeno (ERO) se detectaron mediante un equipo de detección de glutamina, un equipo de detección de GSH y un equipo de detección de ERO, respectivamente. RESULTADOS: Los resultados del ensayo de MTT mostraron que el topotecán tenía un efecto inhibitorio sobre la proliferación celular y que inducía la apoptosis en las estirpes celulares L02 y HepG2. El topotecán inhibió la expresión del transportador de glutamina ASCT2 y la absorción de glutamina en las estirpes celulares L02 y HepG2. La absorción de glutamina y el nivel de GSH aumentaron en las estirpes celulares L02 y HepG2 después de la sobreexpresión del ASCT2. El nivel de ERO fue inhibido por la sobreexpresión del ASCT2 tras el tratamiento con topotecán en las estirpes celulares L02 y HepG2. La apoptosis hepatocelular y la inhibición de la proliferación inducidas por el topotecán fueron atenuadas por la sobreexpresión del ASCT2 en las estirpes celulares L02 y HepG2. CONCLUSIÓN: La muerte de hepatocitos inducida por el topotecán depende de la regulación descendente del ASCT2, que causa estrés oxidativo al inhibir la producción de GSH

Topotecan induces hepatocellular injury via ASCT2 mediated oxidative stress.

BACKGROUND: Topotecan is an anti-cancer chemotherapy drug with common side effects, including hepatotoxicity. In this study, we aim to investigate the mechanisms of topotecan-induced hepatocellular injury beyond conventional DNA damage. MATERIALS AND METHODS: Methyl Thiazolyl Tetrazolium (MTT) assay was used to detect the inhibitory effect of topotecan on cell proliferation. Western blot was used to detect protein expression. Flow cytometry assay was performed to determine apoptosis rate under topotecan treatment. ASCT2 overexpression was addressed using adenovirus vector. qRT-PCR and western blot assay were used to detect the expression of ASCT2. Glutamine uptake, intracellular glutathione (GSH) and reactive oxygen species (ROS) level were detected by glutamine detection kit, GSH detection kit and ROS detection kit respectively. RESULTS: MTT results showed that topotecan had an inhibitory effect on cell proliferation and induced apoptosis in both L02 and HepG2 cell lines. Topotecan inhibited the expression of glutamine transporter ASCT2 and the uptake of glutamine in both L02 and HepG2 cell lines. The uptake of glutamine and the GSH level was increased in both L02 and HepG2 cell lines after ASCT2 overexpression. The ROS level was inhibited by ASCT2 overexpression upon topotecan treatment in both L02 and HepG2 cell lines. Topotecan-induced hepatocellular apoptosis and proliferation inhibition were attenuated by ASCT2 overexpression in both L02 and HepG2 cell lines. CONCLUSION: Topotecan-induced hepatocytes death is dependent on ASCT2 down-regulation, which causes oxidative stress via inhibiting GSH production.

Effects of Extraction Conditions on Crude Polysaccharides and Antioxidant Activities of the Lion's Mane Medicinal Mushroom, Hericium erinaceus (Agaricomycetes).

Dietary supplements are important to sustain an adequate level of antioxidants to balance reactive oxygen species (ROS) in vivo. Hericium erinaceus is one of the rare wood-rotting mushrooms with polysaccharides, which play antioxidant roles in multiple physiological systems of the organism. Can higher polysaccharide content yield higher antioxidant activity? The research on this is scarce. Therefore, the influence of extraction conditions on contents and antioxidant activities of polysaccharide from H. erinaceus was investigated by response surface methodology. Three main independent variables (extraction temperature, time, solid-liquid ratio) were taken into consideration. The extraction and the antioxidant activities were optimized using a Box-Behnken design. Interestingly, the effects of each factor were personalized. Extraction temperature was the dominant factor influencing the polysaccharide contents and antioxidant activities. In addition, the optimal condition to obtain the highest yield of polysaccharide was not in accord with the optimal condition to maximize the antioxidant activities. The effects of every extraction factor on antioxidant activities were various, probably because different components obtained under different extraction conditions had diverse antioxidant mechanisms. This study will help researchers to focus more on the effective components and their antioxidant abilities, rather than blindly pursue the yields of total polysaccharides.

[Experimental Study on Paeoniflorin Inhibiting mTOR Signaling Pathway in Adjuvant Arthritis Rats].

OBJECTIVE: To study the effect of paeoniflorin (PF) on mTOR signal in synovial fibroblast-like synoviocytes (FLS) in rats with adjuvant arthritis. METHODS: AA model rats were prepared by complete Freun's adjuvant injection in foot-plantar, the PF was injected to rats in AA + PF 100 µg / mL group, AA + PF 200 µg / mL group and AA + PF 400 µg / mL group by the tail vein injection at the dose of 0.1 mL/200 g body mass, and the effects of three doses of PF on arthritis scores in AA rats were studied. The modeling rats and control rats were sacrificed at 28 d after modeling, then the synovium was separeated from rat articular, the FLS were cultured. The effect of PF on the expression of mTOR and MMP3 in AA FLS was detected by the real time qPCR. The effect on the cytokine IL-1, IL-6 was detected by ELISA, and the Western blot was used to investigate the role of PF in the mTOR phosphorylation. Furthermore, FLS were transfected with mTOR vectors, and the effect of mTOR overexpression on the PF roles was detected by real time qPCR and ELISA. RESULTS: The tail vein injection of PF can significantly reduce the AA rat arthritis score. Compared with AA group, the expression of mTOR in AA+PF 1 µg/mL, AA+PF 2 µg/mL, AA+PF 4 µg/mL was significantly decreased at 48 h after dosing. Compared with AA group, the relative expression of p-mTOR protein in PF 2 µg/mL group was also decreased. Compared with AA group at 48 h after dosing, the levels of IL-1, IL-6 and MMP3 in AA+PF 1 µg/mL, AA+PF 2 µg/mL, AA+PF 4 µg/ mL were significantly decreased, respectively. Compared with PF 2 µg/mL group, the relative expression of IL-1, IL-6 and MMP3 in PF 2 µg/mL+mTOR vectors was increased. CONCLUSION: PF can significantly inhibit the pathology of AA rats, and its mechanism may be related to the inhibition of mTOR signal in FLS of AA rats.

Secretion expression of human neutrophil peptide 1 (HNP1) in Pichia pastoris and its functional analysis against antibiotic-resistant Helicobacter pylori.

Human neutrophil peptide 1 (HNP1) is a small (3.44 kDa) cationic peptide that is a distinct member of the defensin family. HNP1 plays a crucial role in controlling bacterial infections, particularly by antibiotic-resistant bacteria, through membrane perforation patterns. The structural characteristics of HNP1's three intramolecular disulfide bridges cause difficulty in its synthesis via chemical methods. In this study, bioactive recombinant HNP1 was produced using the Pichia pastoris (P. Pichia) expression system. HNP1 was fused with the polyhedrin of Bombyx mori and enhanced green fluorescent protein (EGFP) to prevent HNP1 toxicity in yeast host cells under direct expression. An enterokinase protease cleavage site (amino acid sequence DDDDK) was designed upstream of the HNP1 peptide to obtain the antibacterial peptide HNP1 with native structure after it was cleaved by the enterokinase. The fusion HNP1 protein (FHNP1) was successfully expressed and had a molecular mass of approximately 62.6 kDa, as determined using SDS-PAGE and Western blot. Then, the recovered FHNP1 was digested and purified; Tricine-SDS-PAGE results showed that HNP1 was successfully released from FHNP1. Functional analysis of induction against antibiotic-resistant Helicobacter pylori (H. pylori) showed that it was challenging for HNP1 to acquire resistance to the antibiotic-resistant H. pylori. Moreover, in vitro studies showed that HNP1 exerted a strong effect against antibiotic-resistant H. pylori activity. Furthermore, the animal model of H. pylori infection established in vivo showed that HNP1 significantly reduced the colonization of antibiotic-resistant H. pylori in the stomach. Our study indicated that this could be a new potential avenue for large-scale production of HNP1 for therapeutic application against the antibiotic-resistant H. pylori infection in humans.

[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.

Fusion expression of the PGLa-AM1 with native structure and evaluation of its anti-Helicobacter pylori activity.

Helicobacter pylori (H. pylori) shows increasingly enhanced resistance to various antibiotics, and its eradication has become a major problem in medicine. The antimicrobial peptide PGLa-AM1 is a short peptide with 22 amino acids and exhibits strong antibacterial activity. In this study, we investigated whether it has anti-H. pylori activity for the further development of anti-H. pylori drugs to replace existing antibiotics. However, the natural antimicrobial peptide PGLa-AM1 shows a low yield and is difficult to separate, limiting its application. A good strategy to solve this problem is to express the antimicrobial peptide PGLa-AM1 using gene engineering at a high level and low cost. For getting PGLa-AM1 with native structure, in this study, a specific protease cleavage site of tobacco etch virus (TEV) was designed before the PGLa-AM1 peptide. For convenience to purify and identify high-efficiency expression PGLa-AM1, the PGLa-AM1 gene was fused with the polyhedrin gene of Bombyx mori (B. mori), and a 6 × His tag was designed to insert before the amino terminus of the fusion protein. The fusion antibacterial peptide PGLa-AM1 (FAMP) gene codon was optimized, and the gene was synthesized and cloned into the Escherichia coli (E. coli) pET-30a (+) expression vector. The results showed that the FAMP was successfully expressed in E. coli. Its molecular weight was approximately 34 kDa, and its expression level was approximately 30 mg/L. After the FAMP was purified, it was further digested with TEV protease. The acquired recombinant antimicrobial peptide PGLa-AM1 exerted strong anti-H. pylori activity and therapeutic effect in vitro and in vivo.

Human Lysozyme Synergistically Enhances Bactericidal Dynamics and Lowers the Resistant Mutant Prevention Concentration for Metronidazole to Helicobacter pylori by Increasing Cell Permeability.

Metronidazole (MNZ) is an effective agent that has been employed to eradicate Helicobacter pylori (H. pylori). The emergence of broad MNZ resistance in H. pylori has affected the efficacy of this therapeutic agent. The concentration of MNZ, especially the mutant prevention concentration (MPC), plays an important role in selecting or enriching resistant mutants and regulating therapeutic effects. A strategy to reduce the MPC that can not only effectively treat H. pylori but also prevent resistance mutations is needed. H. pylori is highly resistant to lysozyme. Lysozyme possesses a hydrolytic bacterial cell wall peptidoglycan and a cationic dependent mode. These effects can increase the permeability of bacterial cells and promote antibiotic absorption into bacterial cells. In this study, human lysozyme (hLYS) was used to probe its effects on the integrity of the H. pylori outer and inner membranes using as fluorescent probe hydrophobic 1-N-phenyl-naphthylamine (NPN) and the release of aspartate aminotransferase. Further studies using a propidium iodide staining method assessed whether hLYS could increase cell permeability and promote cell absorption. Finally, we determined the effects of hLYS on the bactericidal dynamics and MPC of MNZ in H. pylori. Our findings indicate that hLYS could dramatically increase cell permeability, reduce the MPC of MNZ for H. pylori, and enhance its bactericidal dynamic activity, demonstrating that hLYS could reduce the probability of MNZ inducing resistance mutations.

Critical roles of microRNAs in the pathogenesis of systemic sclerosis: New advances, challenges and potential directions.

Systemic sclerosis (SSc) is an autoimmune disease characterized by immune disorders, vascular obliteration, excessive extracellular matrix deposition, skin fibrosis, and further pathological change of internal organs. To date, the exact etiology of this complicated disease remains unknown. Over the past few years, the roles of epigenetic modifications caused by environmental factors have been intensively studied in relation to the disease pathogenesis, and important advances have been made. This review focuses on the new advances of microRNAs (miRNAs) in the field of SSc research, including the upstream regulatory factors of miRNAs, the downstream targets, and the feedback mechanisms between miRNAs and their targets. We also discussed the correlation of miRNAs and DNA methylation, the miRNAs and the gene polymorphism. Overall, the findings presented in this review illustrated how miRNAs play important roles in the pathogenesis of SSc. However, several unanswered questions continue to impede our understanding of this complex disease. Future research should focus on the identification of new biomarkers for early diagnosis and prognosis, which will help us improve the clinical treatment of patients with SSc. In addition, we discussed the challenges of miRNA study in SSc in the future. Since the miRNA injection may be a promising therapeutic approach for SSc treatment, one of the challenges in the future is to evaluate the therapeutic effects of miRNA and anti-miRNAs using SSc model animals. In light of the fact that one miRNA can target many mRNAs, and one mRNA is targeted by many miRNAs, the effect of miRNA changes on other gene expression should be investigated to evaluate the treatment safety of miRNA injection in vivo.

[Effect of Phellinus Polysaccharide on Canonical Wnt Signaling in Synovium in Rats with Rheumatoid Arthritis].

OBJECTIVE: To investigate the role of Phellinus Polysaccharide (FPS) in activating canonical Wnt signaling in rats with rheumatoid arthritis (RA). METHODS: Male SD rats were randomly divided into three groups (normal group, RA model group and PPS treated RA group), each with 10 rats. The RA model rats were prepared through intradermal injection of 0. 1 mL complete Freund's adjuvant into the right rear toes of the rats. The PPS treated RA rats were given 50 mg/kg PPS by gavage eight days after the introduction of RA. All rats were evaluated with arthritis score and paw swelling score at day 16, 20, 24, 28, 32. At day 28, the expressions of fibronectin gene, Wnt signal pathway negative regulation gene SFRP1,2 and Wnt key gene ß-catenin, C-myc, and ccndl were detected by real time qPCR. RESULTS: PPS significantly reduced the arthritis score and paw swelling score of RA model rats. Lower levels of expression of SFRP1, 2 and higher levels of expression of ß-catenin, C-myc, ccndl and fibronectin were found in the RA model rats compared with the normal controls. PPS increased the expression of SFRP1, 2 and decreased the expression of ß-catenin, C-myc, ccndl and fibronectin in the RA rats. CONCLUSION: PPS has significant therapeutic effect on RA model rats through inhibiting canonical Wnt signaling.

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.

Design of signal peptide bombyxin and its effect on secretory expression efficiency and levels of Helicobacter pylori urease subunit B in silkworm cells and larvae

This study employed a Bac-to-Bac/Bombyx mori bioreactor to mass-produce immunogenic urease subunit B (UreB) from Helicobacter pylori. The signal peptide bombyxin from B. mori was used to promote secretory expression to improve expression levels and was designed and integrated into the UreB gene to generate the Bacmid/BmNPV/(signal peptide)-UreB baculovirus expression system. To determine whether the bombyxin signal peptide resulted in secretory expression of recombinant UreB (rUreB) and to determine the secretory efficiency, we tested the secretory expression level of rUreB in Bm5 cells using ELISA. To further investigate whether secretory expression affected cell viability, cells were evaluated using 0.4% trypan blue staining, and Bacmid/BmNPV/UreB without the signal peptide served as a control. The above recombinant bacmid constructs were injected to silkworm larvae, and the secretory expression level of rUreB was detected using SDS-PAGE and semi-quantitative western blot analysis. The results indicated that the bombyxin signal peptide directed the secretory expression of rUreB and that this expression improved the viability of Bm5 cells. Moreover, the results showed that the expression level of rUreB was 1.5 times higher with the Bacmid/BmNPV constructs containing the bombyxin signal sequence than those without the signal sequence. These results demonstrate that secretory expression can enhance rUreB expression levels and is likely to aid in the large-scale expression and yield of rUreB in silkworm larvae.

[Flavonoids of Echinps latifolius suppress Wnt signaling in adjuvant arthritis rats].

The role of flavonoids of Echinps latifolius (FELT) in Wnt signaling was investigated in adjuvant arthritis (AA) rats. The therapeutic effects of FELT on AA rats were detected by rat arthritis score and MTT. The effect of FELT gavage treatment on the Wnt signaling key gene ß-catenin, C-myc and cyclin D1 in synovium from AA rats was detected by Real-time qPCR, and the effects of FELT gavage treatment on the upstream negative regulation gene SFRP 1,2,4,5 in synovium from AA rats were detected by Real-time qPCR. The results showed that FELT gavage treatment significantly inhibited arthritis score and MTT values in AA rats, significantly inhibited the expression of the Wnt signaling gene ß-catenin, C-myc and cyclin D1, significantly up-regulated the expression of the up- stream negative regulation gene SFRP 1,2,4. FELT has a better therapeutic effect for AA rats.

[Treatment of rheumatoid arthritis with flavonoids of Echinps latifolius Tausch in rat model].

OBJECTIVE: To investigate the effect of Flavonoids extracted from Echinps latifolius Tausch(FELT) on rheumatoid arthritis (RA) in rat model. METHOD: Fifty SD rats were randomly divided into model group, control group, and low, medium, and high-dose FELT groups (n=10 in each group). Complete Freund's adjuvant (0.1 mL) was used to induce RA in rats. FELT in doses of 50 mg/kg, 100 mg/kg, 150 mg/kg was given to rats in low, medium and high-dose FELT groups by gavage, and same volume of PBS was given to rats in control group. The arthritis score and the paw swelling score were measured to evaluate the therapeutic effect of FELT. Real time qPCR was used to detect the mRNA expression of fibronectin and MMP3 in synovial tissue and the mRNA expression of caspase 3, Bcl-2 and Bcl-2 associated X protein (Bax) in fibroblast-like synoviocytes (FLS). RESULTS: The arthritis score and the paw swelling score were significantly decreased in three FELT groups compared to RA model rats (P <0.05). The relative expression levels of FN and MMP3 mRNA in synovium of three FELT-treatment groups were significantly lower than those in model group (1.80, 1.76 and 1.67 vs 2.53; 1.69, 1.46 and 1.45 vs 2.67, respectively, all P <0.05). The relative expression levels of Bax and caspase 3 mRNA in FLSs of three FELT groups were higher than those in model group (0.56, 0.58 and 0.60 vs 0.30; 0.54, 0.56 and 0.59 vs 0.29, respectively, all P <0.05); while the relative expression levels of Bcl-2 mRNA in FELT groups were lower than that in model group (2.20, 2.08 and 2.08 vs 4.04, respectively, P <0.05). CONCLUSION: FELT may inhibit the synovium proliferation in RA model rats through promoting the FLS apoptosis.

[Pulchinenoside inhibits the fibroblast-like synoviocytes apoptosis in adjuvant arthritis rats].

OBJECTIVE: To explore the eff ect of pulchinenoside (PULC) on fi broblast-like synoviocytes (FLS) apoptosis in adjuvant arthritis (AA) rats. METHODS: A total of 60 SD rats were randomly divided into 8 groups: A normal control group, an AA group, a low PULC group (50 mg/kg), a middle PULC group (100 mg/kg) or a high PULC group (150 mg/kg) and an ibuprofen (8 mg/kg) group (n=10 per group). FLS from the AA rats was cultured. The expression of Bcl-2, Bax, caspase-3 and the FLS proliferation were detected by the real time qPCR and MTT, respectively. The expression of IL-6 and IL-8 in culture medium was detected by ELISA. RESULTS: Compared with the AA group, the Bcl-2 expression was down-regulated (all P<0.05), the Bax and caspase-3 expression was up-regulated (all P<0.05), and the FLS proliferation was inhibited (all P<0.05). The IL-6 and IL-8 expression was suppressed in the FLS in the PULC groups at different dosages (all P<0.05) as well as in the ibuprofen group (P<0.05). CONCLUSION: PULC may inhibit the FLS proliferation in AA rats by increase in FLS apoptosis.

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.

[Effect of pulchinenoside on FZD8 expression of adjuvant arthritis rats].

To study the effect of pulchinenoside (PULC) on the Frizzled (FZD) expression of adjuvant arthritis ( AA) rats. AA rats were prepared through the toe injection with complete Freund's adjuvant to culture fibroblast-like synoviocytes (FLS). The effect of the oral administration with PULC on the FZD8 expression was detected by the real time qPCR. The effect of FZD8 knockout on the expressions of IL-1, IL-6, IL-8 were detected by MTT and ELISA. The role of miR-375 in the abnomal expression of FZD8 was detected by the real time qPCR. The results showed signfiicant decrease in the FZD8 expression among AA rats, FLS proliferation ater FZD8 knockout and IL-1, IL-6, IL-8 expressions and notable increase in miR-375 expression after the oral administration with PULC. The up-regulated miR-375 expression can inhibit the FZD8 expression. PULC may inhibit the FZD8 expression by up-regulating the miR-375 expression.

[Pulchinenoside control MeCP2 expression in FLS from RA model rats].

The role of pulchinenoside (PULC) in the regulation of MeCP2 expression was investigated in RA model rats. Adjuvant arthritis rats were used as RA model rats, and fibroblast-like synoviocytes (FLS) from the RA model rats were cultured. The effect of 100 mg x kg(-1) PULC gavage treatment on the MeCP2 expression and the effect of MeCP2 siRNA on the expression of SFRP2 and ß-catenin were detected by real time qPCR and Western blotting. The role of PULC in the FLS proliferation was detected by MTT. The results showed that the MeCP2 expression was down-regulated, the SFRP2 expression was up-regulated and the FLS proliferation was inhibited in FLS after therapy. MeCP2 siRNA significantly inhibited the MeCP2 expression, up-regulated the SFRP2 expression and inhibited the ß-catenin expression in FLS from RA model rats. PULC may increase the SFRP2 expression, inhibit the Wnt signaling and inhibit the FLS proliferation in FLS from the RA model rats by inhibiting the MeCP2 expression.