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.

[Qingshen Granules-medicated serum reduces transdifferentiation of NRK-52E cells by miR-23b-5p-mediated activation of the Nrf2 pathway].

OBJECTIVE: TTo investigate the targeted regulation of the Nrf2 pathway by miR-23b-5p in transdifferentiation of rat renal tubular epithelial NRK-52E cells induced by transforming growth factor ß1(TGF-ß1)and the effect of Qingshen Granulesmedicated serum for alleviating transdifferentiation of NRK-52E cells. METHODS: NRK-52E cells with TGF-ß1-induced transdifferentiation were transfected with miR-23b-5p mimic, miR-23b-5p inhibitor or the negative control(NC)siRNA and then treated with of Qingshen Granules-medicated serum.CCK8 assay was used to detectthe changes in viability of NRK-52E cells.The targeting relationship between miR-23b-5p and Nrf2 was verified using a dual luciferase reporter gene assay.The expressions of Nrf2, Keap1 and α-SMA mRNAs and proteins in the treated cells were detected with RT-qPCR and Western blotting, and ROS production in the cells was detected with flow cytometry. RESULTS: Transfection of NRK-52E cells with miR-23b-5p mimic significantly increased the expression of Nrf2 mRNA, while inhibition of miR-23b-5p obviously lowered Nrf2 mRNA in the cells.Rno-miR-23b-5p significantly down-regulated the luciferase activity of Rno-Nrf2-wt but not that of Rno-Nrf2-mu(P<0.05).Treatment with TGF-ß1 significantly decreased the expressions of miR-23b-5p and Nrf2 and increased the expressions of Keap1, α-SMA and ROS in NRK-52E cells(P<0.05), and these changes were obviously ameliorated by treatment with 20% Qingshen Granules-medicated serum for 24 h.Transfection of the cells with miR-23b-mimic significantly decreased the expressions of Keap1, α-SMA and ROS(P<0.05), which were further decreased by treatment with the medicated serum(P<0.05). CONCLUSION: Qingshen Granules-medicated serum reduces transdifferentiation of NRK-52E cells via miR-23b-5pmediated activation of the Nrf2 pathway.

Qingshen granule-medicated serum attenuates high glucose-induced epithelial-mesenchymal transition of HK-2 cells via inhibiting oxidative stress-mediated NF-κB signaling pathway / 中国病理生理杂志

AIM:To investigate whether Qingshen granules(QSG)-medicated serum inhibits oxidative stress-mediated NF-κB signaling pathway and attenuates epithelial-mesenchymal transition(EMT)of human proximal tubule epi?thelial HK-2 cells induced by high glucose. METHODS:The active components in QSG were analyzed by HPLC. The HK-2 cells were randomly divided into control group,mannitol group,high glucose group,low-dose QSG group,medium-dose QSG group,high-dose QSG group and pyrrolidine dithiocarbamate(PDTC)group. The morphological changes of the cells were observed by inverted phase contrast microscopy. MTT assay was used to detect the cell viability. Flow cytometry was used to detect the content of reactive oxygen species(ROS)in HK-2 cells. ELISA was used to detect the content of malondi?al dehyde(MDA)and the activity of superoxide dismutase(SOD). The DNA binding activity of nuclear factor-κB(NF-κB) p65 in HK-2 cells was detected by electrophoretic mobility-shift assay(EMSA). The protein expression of NF-κB p65, phosphorylated inhibitor of kappa B alpha(p-IκBα),inhibitor of kappa B kinase alpha(IKKα),monocyte chemoattractant protein-1(MCP-1)and intercellular adhesion molecule-1(ICAM-1)in HK-2 cells was detected by Western blot. Immuno?fluorescence staining was used to detect NF-κB p65 andα-smooth mucle actin(α-SMA)protein expression in HK-2 cells. RESULTS:Chlorogenic acid,berberine hydrochloride,plantamajoside,6,7-dimethoxycoumarin,epiberberine,copti?sine,lithospermicacid B,palmatine,leonurine hydrochloride,rheic acid and tanshinone ⅡA in QSG were preliminarily de?termined by HPLC. Compared with control group,the levels of ROS and MDA in HK-2 cells induced by high glucose in?creased(P<0. 05),while the activity of SOD decreased(P<0. 05). The protein levels of NF-κB p65,p-IκBα,IKKα, MCP-1,ICAM-1 andα-SMA were increased(P<0. 05). After intervened by QSG-medicated serum,the levels of ROS and MDA were decreased(P<0. 05),while the activity of SOD was increased(P<0. 05). The protein levels of NF-κB p65,p-IκBα,IKKα,MCP-1,ICAM-1 andα-SMA were decreased(P<0. 05). CONCLUSION:QSG-medicated serum inhibited oxidative stress-mediated NF-κB signaling pathway,thus attenuating the EMT of HK-2 cells induced by high glucose.

[Effect of Qingshen Granules on inflammation/hepcidin axis and iron metabolism in patients with renal anemia: a single-center, randomized controlled trial].

OBJECTIVE: To evaluate the therapeutic effect of Qingshen Granules on renal anemia in patients with damp-heat syndrome and explore the mechanisms in light of inflammation/hepcidin axis and iron metabolism. METHODS: Sixty patients with renal anemia and dampness-heat syndrome were randomized into control group (n=30) and treatment group (n=30). All the patients were given routine treatment, and the patients in the treatment group received additional treatment with Qingshen Granules (3 times a day). After 12 weeks of treatments, the patients were examined for changes in the integral value of TCM syndrome, serum creatinine (Scr), glomerular filtration rate (eGFR), hemoglobin (HGB), hematocrit (HCT), red blood cell (RBC) count, interleukin-6 (IL-6), hypersensitive C-reactive protein (hs-CRP), ferritin, growth differentiation factor-15 (GDF-15), serum iron (SI), total iron binding capacity (TIBC), transferrin saturation (TAST), soluble transferrin receptor (sTfR) and ferritin levels. RESULTS: After the treatment, the scores of TCM syndrome were significantly improved in the treatment group and were better than those in the control group (P=0.000). Scr and eGFR were improved in both groups after the treatment. The levels of HGB, HCT and RBC were all improved in the two groups after treatment, and the improvements were more obvious in the treatment group (P=0.002, 0.002, and 0.017, respectively). The levels of IL-6, hs-CRP, hepcidine and GDF-15 were all lowered in the two groups after the treatment, and they were all significantly lower in the treatment group than in the control group (all P=0.000). The treatments increased the levels of SI and TAST in both of the groups, and compared with those in control group, the levels of TIBC, sTfR and ferritin were significantly lowered in the treatment group after the 12-week treatment (P=0.000). CONCLUSIONS: Qingshen granules can effectively improve renal anemia in patients with damp-heat syndrome possibly by improving iron metabolism through alleviation of inflammation and reduction of hepcidine level.

Effect of Qingshen Granules on inflammation/hepcidin axis and iron metabolism in patients with renal anemia: a single-center, randomized controlled trial / 南方医科大学学报

OBJECTIVE@#To evaluate the therapeutic effect of Qingshen Granules on renal anemia in patients with damp-heat syndrome and explore the mechanisms in light of inflammation/hepcidin axis and iron metabolism.@*METHODS@#Sixty patients with renal anemia and dampness-heat syndrome were randomized into control group (=30) and treatment group (=30). All the patients were given routine treatment, and the patients in the treatment group received additional treatment with Qingshen Granules (3 times a day). After 12 weeks of treatments, the patients were examined for changes in the integral value of TCM syndrome, serum creatinine (Scr), glomerular filtration rate (eGFR), hemoglobin (HGB), hematocrit (HCT), red blood cell (RBC) count, interleukin-6 (IL-6), hypersensitive C-reactive protein (hs-CRP), ferritin, growth differentiation factor-15 (GDF-15), serum iron (SI), total iron binding capacity (TIBC), transferrin saturation (TAST), soluble transferrin receptor (sTfR) and ferritin levels.@*RESULTS@#After the treatment, the scores of TCM syndrome were significantly improved in the treatment group and were better than those in the control group (=0.000). Scr and eGFR were improved in both groups after the treatment. The levels of HGB, HCT and RBC were all improved in the two groups after treatment, and the improvements were more obvious in the treatment group (=0.002, 0.002, and 0.017, respectively). The levels of IL-6, hs-CRP, hepcidine and GDF-15 were all lowered in the two groups after the treatment, and they were all significantly lower in the treatment group than in the control group (all =0.000). The treatments increased the levels of SI and TAST in both of the groups, and compared with those in control group, the levels of TIBC, sTfR and ferritin were significantly lowered in the treatment group after the 12-week treatment (=0.000).@*CONCLUSIONS@#Qingshen granules can effectively improve renal anemia in patients with damp-heat syndrome possibly by improving iron metabolism through alleviation of inflammation and reduction of hepcidine level.

Effects of Qingshen granules on Janus Kinase/ signal transducer and activator of transcription signaling pathway in rats with unilateral ureteral obstruction.

OBJECTIVE: To investigate the effects of Qingshen granules (QSG) on janus kinase/ signal transducer and activator of transcription (JAK/STAT) signaling pathway in a rat model of unilateral ureteral obstruction (UUO). METHODS: Sixty male Sprague-Dawley rats were randomly divided into six groups, with 10 animals in each group: the untreated sham-operated normal control group; the untreated UUO model control group, the high dose QSG-treated (16 g?kg-1?d-1) UUO group; the medium dose QSG-treated (8 g?kg-1?d-1) UUO group; the low dose QSG-treated (4 g•kg-1•d-1) UUO group; and the valsartan-treated group (20 mg•kg-1•d-1). The two untreated control groups received physiological saline (1 mL/100 g per day). All the rats were sacrificed after a 4-week course of treatment. Serum creatinine and leptin; protein expressions of leptin receptor (OB-R), p-JAK2, p-STAT3, nuclear factors-kBp6 (NF-kBp65), and monocytechemotatic protein-1 (MCP-1); mRNA of JAK2, STAT3, calcium-dependent adhesion (E-cadherin), alpha- smooth muscle actin (¦Á-SMA) in the kidney tissues; and the expressions of type ¢ô collagen (Col-¢ô and fibronectin (FN) and the pathomorphology in kidney tissues were treated. RESULTS: Compared with the normal group, the BUN, Scr, and serum leptin levels and the expressions of MCP-1, p-JAK2, p-STAT3, NF-kBp65 and OB-R in renal tissues, and the mRNA expressions of leptin, JAK2 protein, STAT3 protein, ¦Á-SMA protein in model group were significantly higher (P < 0.01) in the UUO model group. These parameters were significantly reduced in all the QSG-treated groups and the valsartan-treated group than the UUO model group (P < 0.05 or P < 0.01), with the lowest levels found in the medium dose QSG-treated group (P < 0.05). However, the expression levels of E-cadherin, FN, and Col-¢ô in the renal tissues were contrary to the expressions described above. Severe pathological injury was evident in the renal tissues of UUO model rats, which was alleviated in the QSG-treated and valsartan-treated groups, with the least damage found in the medium dose QSG-treated group. CONCLUSION: Our data suggest that the leptin-mediated JAK/STAT signaling pathway is involved in the process of renal interstitial fibrosis in UUO rats. QSG inhibited the activity of the signaling pathway, reduced the activity of NF-kB and inflammatory effect, and the transdifferentiation in the renal tubular epithelial cells. Treatment with QSG may delay the renal fibrosis and protect the renal function from damage following UUO in rats.

Intervention effect of Qingshen Granules on oxidative stress in patients with chronic renal failure of damp-heat syndrome / 中成药

AIM To observe the changes of serum ROS,MDA,SOD levels in chronic renal failure (CRF) patients with damp-heat syndrome and to explore the intervention effect of Qingshen Granules (Hedyotis diffusae Herba,Salviae miltiorrhizae Radix et Rhizoma,Artemisiae scopariae Herba,etc.) on oxidative stress.METHODS Seventy cases of CRF patients with damp-heat syndrome were randomly and equally divided into control group and treatment group.Sixty cases completed the study,thirty-one cases in the treatment group,twenty-nine cases in the control group.Twenty other cases served as the normal group.The treatment group and the control group were given the basic treatment of western medicine and retention enema of TCM.The treatment group was added Qingshen Granules for eight weeks.The levels of U-Pro/24 h,Scr,BUN,estimated glomerular filtration rate (eGFR),serum ROS,MDA and SOD were measured before and after the treatment,and were compared with the normal group.RESULTS Both total effective rates on disease and on traditional Chinese medicine syndrome in the treatment group were 83.87％,significantly higher than those in the control group (51.72％ and 48.28％),respectively.The levels of U-Pro/24 h,Scr and BUN were obviously lower in the treatment group after the treatment,and eGFR was obviously higher as compared with those in the control group.Before the treatment,the levels of ROS and MDA in the treatment and control groups were significantly higher than those in the normal group,and the level of SOD was significantly lower;the levels of ROS and MDA were reduced in the treatment group after the treatment,and the level of SOD was increased;these changes in the treatment group were more significant than those in the control group.CONCLUSION Qingshen Granules can ameliorate clinical symptoms in CRF patients with damp-heat syndrome,decrease the levels of U-Pro/24 h,Scr and BUN,increase eGFR level,inhibit oxidative stress,and enhance antioxidant capacity,ultimately delay the development of renal fibrosis.

Effects of Qingshen Granules on Life Quality of Chronic Kidney Disease Patients with Damp-heat Syndrome / 中国中医药信息杂志

Objective To observe the improvement effects of Qingshen Granule on the life quality of chronic kidney disease (CKD) patients with damp-heat syndrome. Methods The CKD patients with damp-heat syndrome were randomly divided into experimental group and control group. The control group was treated with Western medicine, and the treatment group was treated with Qingshen Granules additionally for 12 weeks. SCr and eGFR were observed before and after treatment, and the life quality was investigated by KDQOL-SFTM1.3. Results Actually 156 cases were completed, including 77 cases in the treatment group and 79 cases in the control group. The total rate of curative effect was 81.82% (63/77) in teatment group, and (63.29%, 50/79) in control group, with significent difference (P<0.05). Multivariate linear regression analysis indicated that gender, education level, TCM syndrome score, CKD stage, eGFR and Hb level could affect the life quality. The scores of KDQOL-SFTM and its containing the MOS item short from health survey (SF-36) and kidney disease targeted areas (KDTA) were significantly improved after treatment in experimental group (P<0.05), while the scores in control group were with no significant improvement. The improving effects of PCS, and SPL, SLEEP, PS in KDTA in treatment group were significantly superior to those in control group (P<0.05). The scores of life quality in effective and stable cases increased significantly after treatment compared with before treatment (P<0.05). Conclusion Qingshen Granules can effectively improve the life quality of CKD patients with damp-heat syndrome, and the effect is not dependent on the improvement of laboratory indexes (renal function).

Study on Quality Standard of Qingshen Granules / 中国中医药信息杂志

Objective To establish the quality standard of Qingshen Granules. Methods Thin-layer chrmatography was used to identify Rheum officinale Baill., Hedyotis Diffusa Wild. and Coptis chinensis Franch.. The contents of rhein, emodin and chrysophanol were determined by HPLC. The HPLC consisted of Kromasil C18 (4.6 mm×250 mm, 5 μm), methanol-0.1% phosphonic acid (70∶30) as mobile phase, flow rate of 1.0 mL/min, detection wavelength at 245 nm, and the column temperature was 30 ℃. Results The results of TLC showed that relevant spots were clear without interference against the negative sample. The calibration curves for rhein, emodin and chrysophanol were found to be linear within the range of 0.003 3-0.42 μg (r =0.999 9), 0.008 0-0.51 μg (r=1.000 0), 0.009 9-0.32 μg (r=1.000 0), respectively. The average recoveries were 98.84%, 99.04% and 100.35% with RSD of 1.30% (n=6), 1.34% (n=6) and 1.89% (n=6), respectively. Conclusion The methods are accurate and quick in qualitative identification and quantitative assay, and can be used for the quality control of Qingshen Granules.