Rongguo Fu

Activation of AMPK by metformin inhibits TGF-β-induced collagen production in mouse renal fibroblasts.

AIMS To clarify whether activation of adenosine monophosphate-activated protein kinase (AMPK) by metformin inhibits transforming growth factor beta (TGF-β)-induced collagen production in primary cultured mouse renal fibroblasts and further to address the molecular mechanisms. MAIN METHODS Primary cultured mouse renal fibroblasts were stimulated with TGF-β1 and the sequence specific siRNA of Smad3 or connective tissue growth factor (CTGF) was applied to investigate the involvement of these molecular mediators in TGF-β1-induced collagen type I production. Cells were pre-incubated with AMPK agonist metformin or co-incubated with AMPK agonist metformin and AMPK inhibitor Compound C before TGF-β1 stimulation to clarify whether activation of AMPK inhibition of TGF-β1-induced renal fibroblast collagen type I expression. KEY FINDINGS Our results demonstrate that TGF-β1 time- and dose-dependently induced renal fibroblast collagen type I production; TGF-β1 also stimulated Smad3-dependent CTGF expression and caused collagen type I generation; this effect was blocked by knockdown of Smad3 or CTGF. Activation of AMPK by metformin reduced TGF-β1-induced collagen type I production by suppression of Smad3-driven CTGF expression. SIGNIFICANCE This study suggests that activation of AMPK might be a novel strategy for the treatment of chronic kidney disease (CKD) partially by inhibition of renal interstitial fibrosis (RIF).

Sorafenib ameliorates renal fibrosis through inhibition of TGF-β-induced epithelial-mesenchymal transition.

Objective This study was to investigate whether sorafenib can inhibit the progression of renal fibrosis and to study the possible mechanisms of this effect. Methods Eight-week-old rats were subjected to unilateral ureteral obstruction (UUO) and were intragastrically administered sorafenib, while control and sham groups were administered vehicle for 14 or 21 days. NRK-52E cells were treated with TGF-β1 and sorafenib for 24 or 48 hours. HE and Masson staining were used to visualize fibrosis of the renal tissue in each group. The expression of α-SMA and E-cadherin in kidney tissue and NRK-52E cells were performed using immunohistochemistry and immunofluorescence. The apoptosis rate of NRK-52E cells was determined by flow cytometry analysis. The protein levels of Smad3 and p-Smad3 in kidney tissue and NRK-52E cells were detected by western blot analysis. Results HE staining demonstrated that kidney interstitial fibrosis, tubular atrophy, and inflammatory cell infiltration in the sorafenib-treated-UUO groups were significantly decreased compared with the vehicle-treated-UUO group (p<0.05). Masson staining showed that the area of fibrosis was significantly decreased in the sorafenib-treated-UUO groups compared with vehicle-treated-UUO group (p<0.01). The size of the kidney did not significantly increase; the cortex of the kidney was thicker and had a richer blood supply in the middle-dose sorafenib group compared with the vehicle-treated-UUO group (p<0.05). Compared with the vehicle-treated-UUO and TGF-β-stimulated NRK-52E groups, the expression of a-SMA and E-cadherin decreased and increased, respectively, in the UUO kidneys and NRK-52E cells of the sorafenib-treated groups (p<0.05). The apoptotic rate of NRK-52E cells treated with sorafenib decreased for 24 hours in a dose-dependent manner (p<0.05). Compared with the vehicle-treated UUO and TGF-β-stimulated NRK-52E groups, the ratio of p-Smad3 to Smad3 decreased in the sorafenib-treated groups (p<0.05). Conclusion Our results suggest that sorafenib may useful for the treatment of renal fibrosis through the suppression of TGF-β/Smad3-induced EMT signaling.

Activation of PPAR-γ inhibits PDGF-induced proliferation of mouse renal fibroblasts

Recent studies have shown that activation of peroxisome proliferators activated receptor-γ (PPAR-γ) ameliorates renal interstitial fibrosis (RIF) in animal model. Yet, the underlying molecular mechanisms remain still largely unknown. Here, we investigated the hypothesis that activation of PPAR-γ regulates renal remodeling by modulating proliferation of primary cultured renal fibroblasts. In our present study, platelet-derived growth factor-AA (PDGF-AA), a key isoform of PDGF superfamily as mitogen in RIF, was applied to stimulate renal fibroblasts, the selective inhibitor or sequence specific siRNA of PI3K, skp2 or PPAR-γ was used to investigate the involvement of above molecular mediators in PDGF-AA-induced cell proliferation. Our results demonstrate that PDGF-AA induced proliferation of renal fibroblasts by activating PI3K/AKT signaling and resultant skp2 production. Pre-stimulation of cells with rosiglitazone or adenovirus carrying PPAR-γ cDNA (AdPPAR-γ) blocked PDGF-AA-stimulated cell proliferation, this effect was particularly coupled to PPAR-γ inhibition of AKT phosphorylation and skp2 expression. Inhibition of PPAR-γ by GW9662 restored the suppression of activated PPAR-γ on phosphorylation of AKT and subsequent skp2 production. Our results indicate that activation of PI3K/AKT signaling and resultant skp2 generation mediated PDGF-induced proliferation of renal fibroblasts. Activation of PPAR-γ inhibited cell proliferation by inhibition of AKT phosphorylation and its down-streams.

Inhibition of the K+ Channel KCa3.1 Reduces TGF-β1-Induced Premature Senescence, Myofibroblast Phenotype Transition and Proliferation of Mesangial Cells

Objective KCa3.1 channel participates in many important cellular functions. This study planned to investigate the potential involvement of KCa3.1 channel in premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells. Methods & Materials Rat mesangial cells were cultured together with TGF-β1 (2 ng/ml) and TGF-β1 (2 ng/ml) + TRAM-34 (16 nM) separately for specified times from 0 min to 60 min. The cells without treatment served as controls. The location of KCa3.1 channels in mesangial cells was determined with Confocal laser microscope, the cell cycle of mesangial cells was assessed with flow cytometry, the protein and mRNA expression of KCa3.1, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) were detected with Western blot and RT-PCR. One-way analysis of variance (ANOVA) and Student-Newman-Keuls-q test (SNK-q) were used to do statistical analysis. Statistical significance was considered at P<0.05. Results Kca3.1 channels were located in the cell membranes and/or in the cytoplasm of mesangial cells. The percentage of cells in G0-G1 phase and the expression of Kca3.1, α-SMA and FSP-1 were elevated under the induction of TGF-β1 when compared to the control and decreased under the induction of TGF-β1+TRAM-34 when compared to the TGF-β1 induced (P<0.05 or P<0.01). Conclusion Targeted disruption of KCa3.1 inhibits TGF-β1-induced premature aging, myofibroblast-like phenotype transdifferentiation and proliferation of mesangial cells.

Tea polyphenols may attenuate the neurocognitive impairment caused by global cerebral ischemia/reperfusion injury via anti-apoptosis

Background/aims: Global cerebral ischemia/reperfusion (GCIR) may incur neurocognitive impairment. Tea polyphenols (TP) have strong anti-oxidant capacity. This study planned to investigate the protective effect of TP against the neurocognitive impairment caused by GCIR and its mechanism. Methods: One-stage anterior approach for cerebral four-vessel occlusion (4VO) was used to construct the GCIR model. Sprague Dawley rats were randomly classified into Sham group, GCIR group, and TP group (n = 50 per group). Besides receiving the same 4VO, the rats in TP group were treated with TP (6.4%) injection from the tail vein 30 minutes before cerebral ischemia. Morris water-maze test was used to evaluate the changes in space recognition and memory and open field activity test to assess the activity and motor function of rats. The cell apoptotic study in hippocampal CA1 region at specified time points (12, 24, 48, and 72 hours after surgery) was carried out by the flow cytometry, histology (hematoxylin and eosin staining), and immunohistochemical (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining) examinations. One-way analysis of variance and least significant difference t-test were used and statistical significance considered at P < 0.05. Results: Compared with the GCIR group, the TP group was significantly attenuated in the impairment of space recognition and memory caused by GCIR and so was the neuronal apoptosis in the hippocampal CA1 region (P < 0.05). Conclusion: TP may attenuate the impairment of space recognition and memory caused by GCIR via anti-apoptosis.

Protective effect of tea polyphenols on the blood-brain barrier

This study was to investigate the protective effects of tea polyphenols on the blood-brain barrier (BBB) of rats with global cerebral ischemia/reperfusion (GCIR) injury. Sprague Dawley rats underwent four-vessel occlusion to construct the model of GCIR. Half an hour before complete occlusion, they were treated with tea polyphenols (TP) (6.4%; 100 or 200 mg/kg) via tail intravenous injection. 24 h after reperfusion, BBB permeability was evaluated by measuring brain water content (BWC) and residual amount of Evan’s blue dye in cerebral tissue. In addition to this, MMP-9 and collagen IV protein expression in cerebral tissue were also detected using immunohistochemistry. ANOVA and SNK-q were used to do statistical analysis. Statistical significance was considered at P<0.05. Compared to the untreated, the TP-treated rats had significantly decreased BWC (P<0.05), decreased residual amount of Evan’s blue dye in cerebral tissue (P<0.05), down-regulated MMP-9 (P<0.05) and up-regulated collagen IV expression in brain tissue (P<0.05). It can be concluded from these findings that TP may reduce the MMP-9 mediated collagen IV degradation caused by GCIR to protect the BBB.

An optimal dose of tea polyphenols protects against global cerebral ischemia/reperfusion injury

Previous studies addressing the protection of tea polyphenols against cerebral ischemia/reperfusion injury often use focal cerebral ischemia models, and the optimal dose is not unified. In this experiment, a cerebral ischemia/reperfusion injury rat model was established using a modified four-vessel occlusion method. Rats were treated with different doses of tea polyphenols (25, 50, 100, 150, 200 mg/kg) via intraperitoneal injection. Results showed that after 2, 6, 12, 24, 48 and 72 hours of reperfusion, peroxide dismutase activity and total antioxidant capacity in brain tissue gradually increased, while malondialdehyde content gradually decreased after tea polyphenol intervention. Tea polyphenols at 200 mg/kg resulted in the most apparent changes. Terminal deoxynucleotidyl transferase-mediated nick end labeling and flow cytometry showed that 200 mg/kg tea polyphenols significantly reduced the number and percentage of apoptotic cells in the hippocampal CA1 region of rats after cerebral ischemia/reperfusion injury. The open field test and elevated plus maze experiments showed that tea polyphenols at 200 mg/kg strengthened exploratory behavior and reduced anxiety of cerebral ischemia/reperfusion injured rats. Experimental findings indicate that tea polyphenols protected rats against cerebral ischemia/reperfusion injury and 200 mg/kg is regarded as the optimal dose.

Uremic anorexia and gastrointestinal motility dysfunction correlate with the changes of ghrelin system in hypothalamus.

Ghrelin can act as a signal for meal initiation and play a role in the regulation of gastrointestinal (GI) motility via hypothalamic circuit. This study investigated the correlation between changes of hypothalamic ghrelin system and GI motility dysfunction and anorexia in rats with chronic renal failure (CRF).

Uremic anorexia and ghrelin expression in the amygdala

BACKGROUND/AIMS Ghrelin can act as a signal for mealtime hunger and meal initiation. Amygdala is indispensable in appetitive behavior motivated by learned emotions. This study was to investigate the alteration of ghrelin in the amygdala of rats with chronic renal failure (CRF) and its relation with uremic anorexia. METHODS SD rats were randomly classified into CRF group and control group (n=16 per group). The CRF model was constructed using 5/6 nephrectomy. When plasma creatinine (PCr) and blood urea nitrogen (BUN) in the CRF group were twice more than the normal level, food intake (g/24h) was measured and then all rats were killed for detection of ghrelin protein expression in the amygdala using immunohistochemical analysis and mRNA expression using RT-PCT. Statistics was conducted with one-way analysis of variance, Student-Newman-Keuls-q test and correlation analysis. RESULTS By the 8th week after the surgery, the BUN and PCr of CRF rats exceeded double the normal level, and their food intake was obviously decreased compared with the controls (P<0.05). The protein and mRNA expression of ghrelin in the amygdala of CRF group were significantly reduced, and there was a positive correlation between this reduction and the decrease in food intake (P<0.05). CONCLUSION The reduction of amygdalas ghrelin in CRF rats may be associated with uremic anorexia.

Resveratrol down-regulates endothelin type B receptors in vascular smooth muscle cells via Sirt1/ERK1/2/NF-кB signaling pathways

ABSTRACT Silent information regulator family protein 1 (Sirt1) has gained attention for protective effects against cardiovasc diseases. Vascular smooth muscle endothelin type B (ETB) receptors are related to the pathogenesis of cardiovascular diseases. Elevated oxidized low‐density lipoprotein (ox‐LDL) is associated with atherosclerosis. This study will investigate whether resveratrol (a Sirt1 activator, Res) is involved in oxidized low density lipoprotein (ox‐LDL)‐mediated‐ regulation of ETB receptors in rat superior mesenteric arteries (SMA). The rat SMA segments were cultured in the presence and absence of ox‐LDL with or without Res and specific inhibitor (U0126) for the extracellular signal‐regulated protein kinase 1 and 2 (ERK1/2) for 24 h. After organ culture, the contractile responses to sarafotoxin 6c (S6c) were studied using a sensitive myograph, and the ETB receptor protein expression was detected using Western blotting. The results showed that Res concentration‐dependently suppressed the ox‐LDL ‐induced up‐regulation of ETB receptors expression and receptor‐mediated vasoconstriction. In addition, these effects could be inhibited by U0126. Furthermore, activity of ERK1/2 phosphorylation and P65 acetylation induced by ox‐LDL were blocked by Res. In conclusion, Res down‐regulated ETB receptors through up‐regulating Sirt1 and followed by ERK1/2/NF‐&kgr;B signaling pathways in the organ culture SMA.