MicroRNA­101 inhibits renal tubular epithelial­to­mesenchymal transition by targeting TGF­ß1 type I receptor.

MicroRNAs (miRNAs/miRs) are key regulators of renal interstitial fibrosis (RIF). The present study was designed to identify miRNAs associated with the development of RIF, and to explore the ability of these identified miRNAs to modulate the renal tubular epithelial­to­mesenchymal transition (EMT) process. To this end, miRNAs that were differentially expressed between normal and fibrotic kidneys in a rat model of mercury chloride (HgCl2)­induced RIF were detected via an array­based approach. Bioinformatics analyses revealed that miR­101 was the miRNA that was most significantly downregulated in the fibrotic renal tissue samples, and this was confirmed by RT­qPCR, which also demonstrated that this miRNA was downregulated in transforming growth factor (TGF)­ß1­treated human proximal tubular epithelial (HK­2) cells. When miR­101 was overexpressed, this was sufficient to reverse TGF­ß1­induced EMT in HK­2 cells, leading to the upregulation of the epithelial marker, E­cadherin, and the downregulation of the mesenchymal marker, α­smooth muscle actin. By contrast, the downregulation of miR­101 using an inhibitor exerted the opposite effect. The overexpression of miR­101 also suppressed the expression of the miR­101 target gene, TGF­ß1 type I receptor (TßR­I), and thereby impaired TGF­ß1/Smad3 signaling, while the opposite was observed upon miR­101 inhibition. To further confirm the ability of miR­101 to modulate EMT, the HK­2 cells were treated with the TßR­I inhibitor, SB­431542, which significantly suppressed TGF­ß1­induced EMT in these cells. Notably, miR­101 inhibition exerted a less pronounced effect upon EMT­related phenotypes in these TßR­I inhibitor­treated HK­2 cells, supporting a model wherein miR­101 inhibits TGF­ß1­induced EMT by suppressing TßR­I expression. On the whole, the present study demonstrates that miR­101 is capable of inhibiting TGF­ß1­induced tubular EMT by targeting TßR­I, suggesting that it may be an important regulator of RIF.

Fuzheng Huayu recipe, a traditional Chinese compound herbal medicine, attenuates renal interstitial fibrosis via targeting the miR-21/PTEN/AKT axis.

OBJECTIVE: MicroRNAs (miRNAs) may be viable targets for treating renal interstitial fibrosis (RIF). Fuzheng Huayu recipe (FZHY), a traditional Chinese compound herbal medicine, is often used in China to treat fibrosis. This study sought to assess the mechanisms through which FZHY influences miRNAs to treat RIF. METHODS: RIF was induced in rats by mercury chloride and treated with FZHY. Hydroxyproline content, Masson's staining and type I collagen expression were used to evaluate renal collagen deposition. Renal miRNA profiles were evaluated using a miRNA microarray. Those miRNAs that were differentially expressed following FZHY treatment were identified and subjected to bioinformatic analyses. The miR-21 target gene phosphatase and tensin homolog (PTEN) expression and AKT phosphorylation in kidney tissues were assessed via Western blotting. In addition, HK-2 human proximal tubule epithelial cells were treated using angiotensin II (Ang-II) to induce epithelial-to-mesenchymal transition (EMT), followed by FZHY exposure. miR-21 and PTEN expressions were evaluated via quantitative reverse transcription-polymerase chain reaction (qRT-PCR), while E-cadherin and α-smooth muscle actin (α-SMA) expressions were assessed by immunofluorescent staining and qRT-PCR. Western blotting was used to assess PTEN and AKT phosphorylation. RESULTS: FZHY significantly decreased kidney collagen deposition, hydroxyproline content and type I collagen level. The miRNA microarray identified 20 miRNAs that were differentially expressed in response to FZHY treatment. Subsequent bioinformatic analyses found that miR-21 was the key fibrosis-related miRNA regulated by FZHY. FZHY also decreased PTEN expression and AKT phosphorylation in fibrotic kidneys. Results from in vitro tests also suggested that FZHY promoted E-cadherin upregulation and inhibited α-SMA expression in Ang-II-treated HK-2 cells, effectively reversing Ang-II-mediated EMT. We also determined that FZHY reduced miR-21 expression, increased PTEN expression and decreased AKT phosphorylation in these cells. CONCLUSION: miR-21 is the key fibrosis-related miRNA regulated by FZHY. The ability of FZHY to modulate miR-21/PTEN/AKT signaling may be a viable approach for treating RIF.

Effects of Shenqi Neijin powder on activation and apoptosis of hepatic stellate cells in rats with hepatic fibrosis.

Traditional herbal medicine is usually administrated according to experiences and practices. We aimed to investigate the anti-fibrotic effects of Shenqi Neijin powder (SQNJP) in hepatic fibrosis rats induced by carbon tetrachloride (CCl4). A total of 32 rats were divided into control group, model group, and SQNJP group. The hydroxyproline content was assayed. Histological features of liver tissues were determined with different staining methods. Western blotting analysis and immuno-fluorescence staining were performed to determine the expression of alpha-smooth muscle actin (α-SMA) and the activation of hepatic stellate cells (HSCs). Serial sections were stained with α-SMA immuno-fluorescence staining and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method (TUNEL) in turn to detect the apoptosis of HSCs. Fatty degeneration, deposition of collagen, and interval of fibers were noticed in rats induced by CCl4. After administration of SQNJP, remarkable decrease of fatty degeneration, deposition of collagen, and hydroxyproline content were noticed. Compared with the model group, significant decrease of α-SMA protein was noticed after administration of SQNJP, and remarkable apoptosis of HSCs was noticed after treating with SQNJP. SQNJP showed anti-fibrotic effects through inhibiting HSCs activation and inducing apoptosis of HSCs.

[Feasibility of monitoring clopidogrel resistance with flow cytometric analysis of platelet vasodilator stimulated phosphoprotein phosphorylation].

OBJECTIVE: To evaluate the feasibility of monitoring clopidogrel resistance with flow cytometric analysis of platelet vasodilator stimulated phosphoprotein (VASP) phosphorylation. METHODS: Twenty patients with coronary heart disease (CHD) and 17 healthy volunteers were examined for platelet aggregation rate and phosphorylation of VASP (calculated as platelet reactivity index, PRI) using traditional optical nephelometry and flow cytometry before and after concurrent therapy of clopidogrel and aspirin. RESULTS: No significant differences were found in PRI between CHD group and healthy control group [(89.45∓5.22)% vs (86.58∓4.35)%] before treatment. The PRI in CHD group was significantly lowered after treatment to (67.66∓19.77)% (P<0.05). Clopidogrel resistance was found in 6 (30%) cases in CHD group by flow cytometric analysis, which showed a higher sensitivity than optical nephelometry (10%). CONCLUSION: Flow cytometric analysis of VASP phosphorylation is a more reliable test to specifically evaluate clopidogrel resistance.

Proteomic analysis of the effect of fuzheng huayu recipe on fibrotic liver in rats.

Hepatic fibrosis is a common pathological process of chronic liver diseases and would lead to cirrhosis, and Fuzheng Huayu (FZHY) is an effective Chinese herbal product against liver fibrosis. This study observes FZHY influence on proteome of fibrotic liver with differential proteomic approach and aims to understand FZHY multiple action mechanisms on liver fibrosis. The liver fibrosis models were induced with intraperitoneal injection of dimethylnitrosamine for 4 weeks in rats and divided into model control (model) and FZHY-treated (FZHY) groups, while normal rats were used as normal control (normal). After model establishment, rats in FZHY groups were administered 4 g/kg wt of FZHY for 4 weeks, and normal and model groups were given the same volume of saline. The liver proteins in the above 3 groups were separated by two-dimensional gel electrophoresis (2-DE), the differentially expressed spots were analyzed and compared between normal and model or model and FZHY groups, and then the proteins were identified with mass spectrum analysis and validated partially with western blot and real-time PCR. 1000~1200 spots were displayed on each 2D gel, and a total of 61 protein spots were found with significant intensity difference between normal control or FZHY and model control. 23 most obviously differential spots were excised, and in-gel digestion and 21 peptide mass fingerprints (PMF) were obtained with MALDI-TOF MS analysis, and 14 proteins were identified through protein database searching. Among 14 differentially expressed proteins, 8 proteins in normal and FZHY groups had the same tendency of differential expression compared with the ones in model group. And one of them, vimentin, was validated by western blot and real-time PCR analyses. Our study reveals 12 proteins responsible for fibrogenesis induced by DMN in rats, and among them, 8 proteins in fibrotic liver were regulated by FZHY, including aldehyde dehydrogenase, vimentin isoform (CRA_b), gamma-actin, vimentin, fructose-bisphosphate aldolase B, aldo-keto reductase, S-adenosylhomocysteine hydrolase isoform, and HSP90. It indicates that the action mechanism of FZHY antiliver fibrosis may be associated with modulation of proteins associated with metabolism and stress response, as well as myofibroblast activation. The study provides new insights and data for exploring the liver fibrogenesis pathophysiology and FZHY action mechanism against liver fibrosis.