Effect of Qingshen granules on miR-23b and PINKl/Parkin pathway in rat NRK-52E cell transdifferentiation model / 中国药理学通报

Aim To investigate the targeting mechanism of miR-23b on PINKl/Parkin pathway in transdifferentiation of NRK-52E cellsinduced by TGF-β1, and to elucidate the intervention mechanism of Qingshen granules drug-containing serum on NRK-52E cell transdifferentiation. Methods Ultra-high performance liquid chromatography ( UPLC ) fingerprinting method was used to analyze Qingshen granules. The NRK-52E transdifferentiation model induced by TGF-β1 was constructed. The NRK-52E cells were divided into simulated no-load control group, miR-23b-5p simulated group, inhibitor no-load control group, and miR-23b-5p inhibitor group, after transfection with siRNA, and the effect of miR-23b-5p on PINK1 expression was ob-served. The NRK-52E cells were then divided into normal group, TGF-(31 group, Qingshen granule group, miR-23 b-mimic group, miR-23 b-mimic group, and miR-23b-mimic + Qingshen granule group. Western blot was used to detect the expression of Pinkl, Parkin, LC3 n, Beclin-1, P62 and a-SMA proteins, and RT- PCR was used to detect the expression of miR-23 b-5p, Pinkl, Parkin, Beclin-1 and a-SMA mRNA in NRK- 52E cells. Dual-Luciferase Reporter gene experiment was used to detect the targeting relationship between miR-23b-5p and PINKL Results UPLC fingerprinting method found 11 active components in Qingshen granules. After overexpression of miR-23b-5p, the expression of PINkl mRNA significantly increased (P 0. 05 ). The experimental results showed that the expressions of miR- 23b-5p, Pinkl, Parkin, Beclin-1, LC3 II and LC3 II/ I ratio in TGF-β1 group were significantly lower than those in normal group, but the expressions of P62 and a-SMA were significantly higher than those in normal group ( P <0.05). The expressions of miR-23 b-5 p, Pinkl, Parkin, Beclin-1, LC3 II and LC3 11/ I ratio in Qingshen granule group and miR-23 b-mimic group were significantly higher than those in TGF-β1 group, and the expressions of P62 and a-SMA were significantly lower than those in TGF-β1 group (P < 0. 05 ). The performance of miR-23 b-mimic + Qingshen granule group was better than that of miR-23 b-mimic group (P < 0. 05 ). Conclusions Qingshen granules can up- regulate the expression of miR-23b-5p in NRK-52E cellsand inhibit the transdifferentiation process of NRK- 52E cells by enhancing the mitochondrial autophagy activity mediated by PINKl/Parkin pathway.

Arbutin isolated from Chinese yam inhibits LPS-induced NRK-52e cell apoptosis by ERp / 中国药理学通报

Aim To investigate the effect of arbutin on apoptosis of NRK-52e cells induced by LPS and the potential mechanism.Methods The model of NRK- 52e cells injury was constructed by LPS, and NRK-52e cells were divided into control, LPS ( 1 mg • L 1 ) , low dose arbutin (LPS, 1 mg • L 1 + arbutin, 5 (xmol • L_l ) , high dose arbutin ( LPS, 1 mg • L 1 + arbutin , 10 (xmol • L 1 ) and its corresponding inhibitor THC group ( 1 (xmol • L 1).The cell viability was detected ; the levels of ROS, apoptosis, Ca~' concentration and mitochondrial membrane potential ( MMP ) were detected by flow cytometry; the levels of key apoptosis proteins were detected by in cell western; the binding activity of arbutin with ER(3 was imitated by molecular docking technology, and verified by in cell western.Results Arbutin could effectively regulate the levels of ROS, Ca"+ , apoptosis proteins and ER(3 in NRK-52e cells induced by LPS and inhibit the de- cline of MMP, which is blocked by estrogen receptor inhibitor THC.In addition, arbutin has good binding activity with ERf}.Conclusion This study confirms that arbutin could inhibit LPS-induced apoptosis of NRK-52e cells through ER£.

A new alkaloid from Rehmanniae Radix Preparata / 药学学报

The chemical constituents of Rehmanniae Radix Preparatawere prepared according to the traditional method of "jiu zheng jiu shai" and investigated using multiple chromatographic methods. Six alkaloids were isolated and their structures were elucidated from spectral data and physicochemical properties, as follows: rehmanniae alkaloid A (4-{[(5-O-á-D-galactopyranosyloxy)methyl]-1H-pyrrole-2-carbaldehyde-1-yl}butyric acidmethyl ester) (1), baimantuoluoamide B (2), capparisine C (3), harman-3-carboxylic acid (4), (2S)-1-[2-(furan-2-yl)-2-oxoethyl]-5-oxopyrrolidine-2-carboxylate (5), and 1-[2-(furan-2-yl)-2-oxoethyl]pyrrolidin-2-one (6). Among them, compound 1 is a new alkaloid. Compounds 2-6 were newly isolated from Rehmannia glutinosa Libosch.The effect of compounds 1-6 on NRK-52e cell injury induced by LPS was investigated. The results show that compounds 1-3 exhibit protective effects against LPS-induced damage to NRK-52e cells.

Expression of miR-207 is up-regulated in renal and urine of rats with renal fibrosis / 基础医学与临床

Objective To investigate the differential expression of miR-207 in urine samples and kidney of rat renal fibrosis (renal fibrosis, RF) model and its function was investigated. Methods The rats were divided into two groups,including one undergoing ligation of the right ureter as UUO rat model,the other just under going the same surgery but no ligation for the ureters and as a shamed rat model.Real-time PCR(RT-qPCR) was used to detect the expression of miR-207 in kidney and urine of rat renal fibrosis model. The function of miR-207 was investigated with transfection of miR-207 mimics and inhibitor to the rat renal tubular epithelial NRK-52E cells. Results miR-207 expression in urine and kidney tissues of rat renal fibrosis model was upregulated(P＜0.05). miR-207 in-creased the expression of gene related to fibrosis in rats renal tubular epithelial NRK-52E cells. Conclusions miR-207 may play an important role in renal fibrosis and provide a potential method for the diagnosis of renal fibrosis.

Effects of BMP-7 on Id2 and E2A expression in NRK-52E cells exposed to high glucose / 中国病理生理杂志

AIM: To investigate the effects of bone morphogenetic protein 7 ( BMP-7 ) on the expression of transcription factor E2A and inhibitor of differentiation 2 (Id2) in the renal tubule epithelial cells(NRK-52E)exposed to high glucose, and to explore its possible mechanism of improving renal tubular fibrosis induced by high glucose.METH-ODS:The NRK-52E cells were divided into control group, high glucose (HG) group and high glucose with different doses of BMP-7 (10μg/L and 20μg/L) group.The cells in HG group and BMP-7 group were cultured for 12 h, 24 h and 48 h. The protein expression of Id2, E2A, E-cadherin,α-smooth muscle actin (α-SMA) and collagen-I was detected by Western blot.In addition, the mRNA expression of Id2 was detected by real-time PCR.RESULTS:Compared with control group, the mRNA and protein levels of Id2 and the protein level of E-cadherin were down-regulated, while the protein levels of E2A,α-SMA and collagen-I were up-regulated in HG group (P<0.05).Compared with HG group, the mRNA and pro-tein levels of Id2 and the protein level of E-cadherin were significantly up-regulated, while the protein expression of E2A,α-SMA and collagen-I was significantly down-regulated in 20 μg/L BMP-7 group ( P<0.05 ) .The correlation analysis showed that the Id2 protein level was negatively correlated with the E2A protein level (P<0.05).CONCLUSION:BMP-7 may intercept the process of renal tubule fibrosis induced by high glucose via promoting the expression of Id2 and inhibi-ting the expression of E2A at protein level.

Effects of propofol, etomidate and midazolam on gap junction function in rats / 中华麻醉学杂志

Objective To evaluate the effects of propofol,etomidate and midazolam on gap junction function in rats in vitro and in vivo.Methods Experiment Ⅰ NRK52E cells were seeded in 6-well plates with the density of 1.0 × 105/ml and randomly divided into 4 groups (n =6 wells each):control group (group C),propofol group (group P),etomidate group (group E) and midazolam group (group M).In group C,NRK52E cells were cultured in DMEM-F12 culture medium containing 10％ fetal bovine serum.In P,E and M groups,propofol,etomidate and midazolam (with the final concentrations of 15,8 and 4μmol/L,respectively) were added to the culture medium,respectively,and the cells were then incubated for 1 h.Parachute dye-coupling assay was used to measure the gap junction function in NRK52E cells.Experiment Ⅱ Twenty-four male Sprague-Dawley rats,aged 2 months,weighing 220-280 g,were randomly divided into 4 groups (n =6 each):propofol group (group P),etomidate group (group E),midazolam group (group M),and control group (group C).In P,E and M groups,propofol 100 mg/kg,etomidate 6 mg/kg and midazolam 5 mg/kg were injected intraperitoneally once a day for 3 consecutive days,respectively.The equal volume of normal saline was given instead in group C.The animals were sacrificed at 30 min after the last administration and the renal cortex was harvested to measure the gap junction function using tissue scrape and load assay.Results Compared with group C,the gap junction function was significantly decreased in P and E groups (P ＜ 0.05),and no significant change was found in the gap junction function in group M (P ＞ 0.05).Conclusion Propofol and etomidate significantly inhibit the gap junction function in NRK52E cells and renal tissues in rats,but midazolam produces no effect.

Diminution of oxalate induced renal tubular epithelial cell injury and inhibition of calcium oxalate crystallization in vitro by aqueous extract of Tribulus terrestris

PURPOSE: Recurrence and persistent side effects of present day treatment for urolithiasis restrict their use, so an alternate solution, using phytotherapy is being sought. The present study attempted to evaluate the antilithiatic properties of Tribulus terrestris commonly called as “gokhru” which is often used in ayurveda to treat various urinary diseases including urolithiasis. MATERIALS AND METHODS: The activity of Tribulus terrestris was investigated on nucleation and the growth of the calcium oxalate (CaOx) crystals as well as on oxalate induced cell injury of NRK 52E renal epithelial cells. RESULTS: Tribulus terrestris extract exhibited a concentration dependent inhibition of nucleation and the growth of CaOx crystals. When NRK-52E cells were injured by exposure to oxalate for 72 h, Tribulus terrestris extract prevented the injury in a dose-dependent manner. On treatment with the different concentrations of the plant, the cell viability increased and lactate dehydrogenase release decreased in a concentration dependent manner. CONCLUSION: The current data suggests that Tribulus terrestris extract not only has a potential to inhibit nucleation and the growth of the CaOx crystals but also has a cytoprotective role. Our results indicate that it could be a potential candidate for phytotherapy against urolithiasis.

Effects of Mercuric Chloride on Gene Expression in NRK-52E Cells

Mercuric chloride, a model nephrotoxicant was used to elucidate time- and dose-dependent global gene expression changes associated with proximal tubular toxicity. Rat kidney cell lines NRK-52E cells were exposed for 2, 6 and 12 hours and with 3 different doses of mercuric chloride. Cell viability assay showed that mercuric chloride had toxic effects on NRK-52E cells causing 20% cell death (IC20) at 40micrometer concentration. We set this IC20 as high dose concentration and 1/5 and 1/25 concentration of LC20 were used as mid and low concentration, respectively. Analyses of microarray data revealed that 738 genes were differentially expressed (more than two-fold change and p<0.05) by low concentration of mercuric chloride at least one time point in NRK-52E cells. 317 and 2,499 genes were differentially expressed at mid and high concentration of mercuric chloride, respectively. These deregulated genes showed a primary involvement with protein trafficking (CAV2, CANX, CORO1B), detoxification (GSTs) and immunity and defense (HMOX1, NQO1). Several of these genes were previously reported to be up-regulated in proximal tubule cells treated with nephrotoxicants and might be aid in promoting the predictive biomarkers for nephrotoxicity.

Effect of losartan on expression of PTHrP and PTH1R induced by high glucose in NRK-52E cells / 中华内分泌代谢杂志

To study the effect of losartan on high glucose up-regulated expression of parathyroid hormone-related peptide (PTHrP) and its type 1 receptor (PTH1R) in NRK-52E cells. The expression of PTHrP and PTH1R were detected by RT-PCR and Western blot in control group (0 mmol/L glucose) ,normal glucose group (5 mmol/ L glucose) ,moderately high glucose group (16.7 mmol/L glucose) ,high glucose group (25 mmol/L glucose) ,and after intervention by 10 μmol/L losartan for 72 h (only Western blot). The expression of PTHrP and PTH1R were up-regulated by high glucose (PTHrP mRNA : 0. 66 ± 0.08, 0. 84 ± 0. 13,1.57 ± 0. 15, and 1.73 ± 0.21 ; PTHrP protein :0.63±0.12,0.68±0.06,1.02±0. 11, and 1.04±0.08 ; PTH1R mRNA :0.26±0. 08,0.28±0.07,2.35± 0. 10,and 2.47±0. 05 ; PTH1R protein:0. 88±0. 05,0. 87±0. 10, 1.05±0. 11, and 1.12±0. 09) ,and losartan inhibited the effects of high glucose (PTHrP 0.74±0. 15, PTH1R 0.98±0.06, both P<0.01). The results suggest that losartan could inhibit the expression of FTHrP and PTH1R induced by high glucose in NRK-52E.

Effects of Endotoxin on Growth Activity and Gap Junction of NRK52E Cells / 中山大学学报(医学科学版)

[Objective]This study was designed to observe the effects of endotoxin(LPS)on the growth activity and gap junction(GJ)of NRK52E cells.[Methods]The NRK52E cells were divided into control group and LPS groups,and the NRK52E cells in LPS groups were treated with LPS 10 ng/mL,50 ng/mL,100 ng/mL,500 ng/mL,and 1 000 ng/mL for 24 h respectively.The NRK52E cells growth activity was measured through MTT method,and the function of gap junction of NRK52E cells was measured through the method of fluoroimmunoassay.The protein expression of connexin 43(Cx43)in control group,LPS 10 ng/mL group,and LPS 100 ng/mL group were also determined by Western blotting.[Results]①Compared with control group and LPS 10 ng/mL group,The NRK52E cells growth activity decreased significantly in LPS 50 ng/mL,100 ng/mL,500 ng/mL,and 1 000 ng/mL groups(P＜0.01).②Compared with control group,the function of GJ decreased significantly in LPS 100 ng/mL,500 ng/mL,and 1 000 ng/mL groups(P＜0.01).③There were negative correlations among the concentration of LPS and NRK52E cells growth activity and GJ function respectively(r=-0.941,-0.872,P＜0.01).④Compared with control group,the protein expression of Cx43 were decreased significantly in LPS 10 ng/mL and 100 ng/mL groups(P＜0.01).[Conclusions]LPS can inhibit the NRK52E cells growth activity in a dose-depend manner.GJ function is one of the mechanisms.

Role of parathyroid hormone-related peptide in transdifferentiation of NRK-52E cells induced by high glucose / 第三军医大学学报

Objective To study the role of parathyroid hormone-related peptide ( PTHrP) in transdif-ferentiation of NRK-52E cells induced by high glucose. Methods Expression of PTHrP and its receptor 1 in NRK-52E cells incubated with 0 ( control group) ,5 ( normal glucose group) and 16. 7mmol/L glucose ( high glucose group) ,respectively,was detected by RT-PCR and Western blotting,respectively. Expression of TGF-?1,MMP2,type Ⅰ collagen and ?-SMA in NRK-52E cells of normal and high glucose groups was detected by Western blotting and ROS was detected by CM2H2DCFDA after 72 h. Results The expression levels of PTHrP,PTHrP receptor 1 ( PTH1R) ,and PTHrP mRNA were higher in high glucose group than in control and normal glucose groups ( P