An integrated strategy by absorbed component characterization, pharmacokinetics, and activity evaluation for identification of potential nephroprotective substances in Zhu-Ling decoction.

An efficient strategy for the identification of potential nephroprotective substances in Zhu-Ling decoction has been established with the integration of absorbed components characterization, pharmacokinetics, and activity evaluation. A qualitative method was developed to characterize the chemical constituents absorbed components in vivo of Zhu-Ling decoction by using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. A quantitative method was established and validated for the simultaneous determination of eight compounds in rat plasma by using ultra-performance liquid chromatography-triple quadruple tandem mass spectrometry. Finally, the nephroprotective activities of absorbed components with high exposure were assessed by cell survival rate, superoxide dismutase, and malondialdehyde activities in hydrogen peroxide-induced Vero cells. As a result, 111 compounds in Zhu-Ling decoction and 36 absorbed components were identified in rat plasma and urine, and poricoic acid A, poricoic acid B, alisol A, 16-oxo-alisol A, and dehydro-tumulosic acid had high exposure levels in rat plasma. Finally, poricoic acid B, poricoic acid A, 16-oxo-alisol A, and dehydro-tumulosic acid showed remarkable nephroprotective activity against Vero cells damage induced by hydrogen peroxide. Besides, superoxide dismutase and malondialdehyde activities were obviously regulated in hydrogen peroxide-induced Vero cells by treatment with the four compounds mentioned above. Therefore, these four compounds were considered to be effective substances of Zhu-Ling decoction due to their relatively high exposure in vivo and biological activity. This study provided a chemical basis for the action mechanism of Zhu-Ling decoction in the treatment of chronic kidney diseases.

[Chemical constituents from green walnut husks and their antitumor activity in vitro].

Fourteen chemical constituents, including 5-hydroxy-4-methoxy-1-tetralone(1), 4,8-dihydroxy-1-tetralone(2), 4,5-dihydroxy-α-tetralone(3), blumenol B(4), dehydrovomifoliol(5), megastigm-5-ene-3,9-diol(6), juglanin B(7), blumenol C(8), loliolide(9), oleracone B(10), syringarsinol(11), pinoresinol(12), methyl 4-hydroxy-3-methoxybenzoate(13), and isovanillic acid(14), were isolated from the dichloromethane fraction of 95% methanol extract of green walnut husks by silica gel and MCI column chromatography, and Pre-HPLC. Their structures were determined by spectroscopic methods, such as NMR, MS and so on. Among them, compounds 1, 4-6, 8-13 were isolated from the green walnut husks for the first time, and compounds 4-6, 8, 10, 12, 13 were isolated from the Juglans genus for the first time. All of isolates were detected their inhibitory activities against HeLa, HGC-27 and Ht-29 cell lines by the MTT assay. The result showed that compounds 2, 3, 7, 9 and 11 exhibited inhibitory activity against the tested cell line. The IC_(50) of 7 were 26.5, 9.0, 25.4 µmol·L~(-1), respectively.

[Clinical analysis of drug-induced Pseudo-Bartter's syndrome: a report of five cases].

OBJECTIVE: To summarize the clinical characteristics and outcomes of Pseudo-Bartter's syndrome and explore its pathogenesis. METHODS: The clinical data of 5 cases of Pseudo-Bartter's syndrome at our ward from May 2008 to December 2010 was analyzed retrospectively. RESULTS: All patients were female. Long-term regimen of purgative or diuretics was prescribed. The clinical features included normotension, hypokalemic alkalosis and activation of renin-angiotensin-aldosterone. The pathological results of 3 cases of kidney biopsy showed the hyperplasia of juxtaglomerular apparatus, thickness of arteriole, infiltration of lymphocytes and monocytes and degeneration of renal tubule. Upon a definitive diagnosis, purgative or diuretics was discontinued and supplement therapy of potassium chloride initiated. The results of laboratory tests reverted to normal ranges within 4 weeks. CONCLUSION: Purgative or diuretics should be prescribed appropriately to avoid the occurrence of Pseudo-Bartter's syndrome.

[Clinical analysis of 6 cases of Bartter syndrome].

OBJECTIVE: To summarize the clinical characteristics of Bartter syndrome and investigate its pathogenesis. METHODS: The clinical data of 6 cases of Bartter syndrome at our hospital from November 2006 to May 2010 were analyzed retrospectively. RESULTS: The onset age of Bartter syndrome was 13-35 years old. The main symptoms included weakness (6/6), paralysis (1/6), numbness (5/6) and tetany (4/6). All patients had normal blood pressure. The biochemical tests showed persistent hypokalemia, metabolic alkalosis (6/6) and hyperreninemia. The pathological examination of deltoid muscle biopsy showed the swelling, degeneration and necrosis of myocytes and the deposition of immunocomplex in myolemma. And the pathological examination of renal biopsy showed the hyperplasia of juxtaglomerular apparatus (5/6) and the deposition of immunocomplex. All symptoms were relieved after a therapy of potassium supplementation or a combination of indomethacin, spironolactone and immunosuppressant. CONCLUSION: When such clinical features as weakness, paralysis, tetany, hypokalemic alkalosis and normotension are encountered, Bartter syndrome should be suspected. Serum electrolytes, blood gas analysis and activation of the renin-angiotensin-aldosterone system should be examined for a definite diagnosis. The treatment of choice includes potassium and magnesium supplementation or in combination with prostaglandin synthetase inhibitor, aldosterone antagonist and immunosuppressant. Immunologic mechanism may participate in the course of Bartter syndrome.

Model of cardiac arrest in rats by transcutaneous electrical epicardium stimulation.

OBJECTIVE: To establish a new model of cardiac arrest (CA) in rats by transcutaneous electrical epicardium stimulation. METHODS: Two acupuncture needles connected to the anode and cathode of a stimulator were transcutaneously inserted into the epicardium as electrodes. The stimulating current was steered to the epicardium and the stimulation was maintained for 3 min to induce CA. Cardiopulmonary resuscitation (CPR) was performed at 6 min after a period of nonintervention. RESULTS: CA was successfully induced in a total of 20 rats. The success rate of induction was 12/20 at the current intensity of 1 mA; and reached 20/20 when the current intensity was increased to 2 mA. After the electrical stimulation, the femoral blood pressure quickly dropped below 25 mmHg and the arterial pulse waveform disappeared. The average time from the electrical stimulation to CA induction was 5.10 (+/-2.81) s. When the electrical stimulation stopped, 18/20 rats had ventricular fibrillation and 2/20 rats had pulseless electrical activity. CPR was performed for averagely 207.4 (+/-148.8) s. The restoration of spontaneous circulation (ROSC) was 20/20. The death rate within 4h after ROSC was 5/20, and the 72-h survival rate was 10/20. There were only two cases of complications, a minor muscle contraction and a minor lung lobe injury. CONCLUSION: The model of CA in rats induced by transcutaneous electrical epicardium stimulation is a stable model that requires low-intensity current and has fewer complications. This model may provide another option for experimental research of CA induced by malignant arrhythmia (especially VF).

Nitroglycerin enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via nitric oxide pathway.

AIM: To investigate the effect of nitroglycerin (NTG) on cell proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells (HBMSC) and its mechanisms. METHODS: Primary HBMSC were cultured in osteogenic differentiation medium consisting of phenol red-free alpha-minimum essential media plus 10% fetal bovine serum (dextran-coated charcoal stripped) supplemented with 10 nmol/L dexamethasone, 50 mg/L ascorbic acid, and 10 mmol/L beta-glycerophosphate for inducing osteoblastic differentiation. The cells were treated with NTG (0.1-10 micromol/L) alone or concurrent incubation with different nitric oxide synthase (NOS) inhibitors. Nitric oxide (NO) production was measured by using a commercial NO kit. Cell proliferation was measured by 5-bromodeoxyuridine (BrdU) incorporation. The osteoblastic differentiation of HBMSC culture was evaluated by measuring cellular alkaline phosphatase (ALP) activity and calcium deposition, as well as osteoblastic markers by real-time RT-PCR. RESULTS: The treatment of HBMSC with NTG (0.1-10 micromol/L) led to a dose-dependent increase of NO production in the conditional medium. The release of NO by NTG resulted in increased cell proliferation and osteoblastic differentiation of HBMSC, as evidenced by the increment of the BrdU incorporation, the induction of ALP activity in the early stage, and the calcium deposition in the latter stage. The increment of NO production was also correlated with the upregulation of osteoblastic markers in HBMSC cultures. However, the stimulatory effect of NTG (10 micromol/L) could not be abolished by either N(G ) -nitro-L-arginine methyl ester, an antagonist of endothelial NOS, or 1400W, a selective blocker of inducible NOS activity. CONCLUSION: NTG stimulates cell proliferation and osteoblastic differentiation of HBMSC through a direct release of NO, which is independent on intracellular NOS activity.