Protective effects of rutin on kidney in type 1 diabetic mice.

Diabetic nephropathy is one the most serious diabetic microangiopathies, which is the main cause of mortality in diabetic patients. Our research investigated the protective effects of rutin on kidney of the type 1 diabetes mice induced by streptozotocin (STZ). The levels of kidney weight index (KWI), postprandial plasma glucose (PPG), creatinine (Cre), blood urine nitrogen (BUN), the activity of super oxide dismutase (SOD), malondialdehyde (MDA) and glutathione per oxidase (GSH-Px) were all measured. The histological morphology of kidney tissues was observed by hematoxylin-eosin (HE) staining, masson staining and electron microscope. The collagen I (COL-I) and transforming growth factor-ß1 (TGF-ß1) levels were estimated by immunohistochemistry, western blot and Real-Time PCR respectively. The results revealed that the levels of SOD and GSH-Px all increased, while the levels of KWI, PPG, Cre, BUN and MDA all decreased in diabetic mice after the rutin treatment for eight weeks. Moreover, the histological morphology of kidney tissues was also improved. Furthermore, the expression of COL-I and TGF-ß1 in kidney tissues increased significantly in the diabetic mice, which were antagonized by the rutin treatment. Together, the result suggested that rutin can improve kidney injury of the type 1 diabetic mice.

Protein kinase C epsilon mediates the inhibition of angiotensin II on the slowly activating delayed-rectifier potassium current through channel phosphorylation.

The slowly activating delayed rectifier K+ current (IKs) is one of the main repolarizing currents in the human heart. Evidence has shown that angiotensin II (Ang II) regulates IKs through the protein kinase C (PKC) pathway, but the related results are controversial. This study was designed to identify PKC isoenzymes involved in the regulation of IKs by Ang II and the underlying molecular mechanism. The whole-cell patch-clamp technique was used to record IKs in isolated guinea pig ventricular cardiomyocytes and in human embryonic kidney (HEK) 293 cells co-transfected with human KCNQ1/KCNE1 genes and Ang II type 1 receptor genes. Ang II inhibited IKs in a concentration-dependent manner in native cardiomyocytes. A broad PKC inhibitor Gö6983 (not inhibiting PKCε) and a selective cPKC inhibitor Gö6976 did not affect the inhibitory action of Ang II. In contrast, the inhibition was significantly attenuated by PKCε-selective peptide inhibitor εV1-2. However, direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) increased the cloned human IKs in HEK293 cells. Similarly, the cPKC peptide activator significantly enhanced the current. In contrast, the PKCε peptide activator inhibited the current. Further evidence showed that PKCε knockdown by siRNA antagonized the Ang II-induced inhibition on KCNQ1/KCNE1 current, whereas knockdown of cPKCs (PKCα and PKCß) attenuated the potentiation of the current by PMA. Moreover, deletion of four putative phosphorylation sites in the C-terminus of KCNQ1 abolished the action of PMA. Mutation of two putative phosphorylation sites in the N-terminus of KCNQ1 and one site in KCNE1 (S102) blocked the inhibition of Ang II. Our results demonstrate that PKCε isoenzyme mediates the inhibitory action of Ang II on IKs and by phosphorylating distinct sites in KCNQ1/KCNE1, cPKC and PKCε isoenzymes produce the contrary regulatory effects on the channel. These findings have provided new insight into the molecular mechanism underlying the modulation of the KCNQ1/KCNE1 channel.

[Effects of Rutin on Myocardial Enzyme and Cardiac Morphology in Diabetic Mice].

OBJECTIVE: To study the effects of rutin on myocardial enzyme in serum and myocardial morphology in diabetic mice induced by streptozotocin (STZ). METHODS: A model of type 1 diabetic mice was established in 48 male kunming mice with daily intraperitoneal injection of streptozotocin (STZ) 62.5 mg/kg for 5 consecutive days. 12 male Kunming mice were selected as normal group randomly. The established successfully diabetic mice were randomly divided into the model group, irbesartan group [45 mg/ (kg?d)], low-, high-dose rutin groups [50, 100 mg/ (kg?d)]. The mice in the normal and the model groups were given sodium carboxymethyl cellulose solution (CMC-Na, 1 g/L) by intragastric administration respectively. After administration for 8 weeks, the levels of creatine kinase (CK), creatine kinase isoenzymes (CK-MB), hydroxybutyrate dehydrogenase (HBDH), and lactate dehydrogenase (LDH) in serum were detected by blood biochemical analyzer. The cardiac myocardial morphology was observed by HE staining, Masson trichrome staining and electron microscope. RESULTS: Compare to the normal group, the levels of the myocardial enzyme in serum evidently increased in the model group (P<0.01); Compare to the model group, the levels of the myocardial enzyme in serum decreased in low-, high-dose rutin groups; The levels of HBDH and LDH declined remarkably in the high-dose rutin group relative to the low-dose group (P<0.05); Compared to the high-dose group, the level of LDH increased in the irbesartan group (P<0.05).Moreover, relative to the model group, the morphology of myocardial tissue, the degree of fibrosis and the ultrastructure of myocardial tissue in mice were significantly improved in low-, high-dose rutin groups, and the effects were more significant in the high-dose rutin group. CONCLUSION: Rutin could decrease the levels of myocardial enzyme in serum in diabetic mice, improve the cardiac cell morphology and alleviate myocardial injury.

YQFM alleviated cardiac hypertrophy by apoptosis inhibition and autophagy regulation via PI3K/AKT/mTOR pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional compound preparation of Chinese medicine, Yiqi Fumai lyophilized injection (YQFM) has protective effects on various cardiac diseases including cardiac hypertrophy, which is the primary cause of arrhythmia. However, the involved mechanism remains unclear. AIM OF THE STUDY: This study was projected to investigate whether YQFM could prevent cardiac hypertrophy and arrhythmia concurrence. MATERIALS AND METHODS: The cardiac hypertrophy rats were established by transverse aortic ligation and the H9c2 hypertrophy cardiomyocyte was induced by angiotensin II (AngII). The electrocardiogram (ECG) was conducted to estimate the arrhythmia occurrence of cardiac hypertrophy rats under isoprenaline (iso) treatment. The cardiac related indicators and histopathology were also detected. The protective effects of YQFM on H9c2 hypertrophy cardiomyocyte were determined by the cell size measurement, apoptosis detection and mitochondrial membrane potential measurement. The cardiac hypertrophy relative proteins (ANP and BNP), autophagy related factors (LC3II, p62 and Beclin-1), apoptosis related markers (p53, caspase 3, Bax and Bcl-2) and the PI3K/AKT/mTOR pathway expressions were all measured by Western blot. RESULTS: YQFM decreased the arrhythmia occurrence and improved cardiac function in cardiac hypertrophy rats. YQFM also reduced the H9c2 cardiomyocyte size and alleviated the cardiomyocyte apoptosis induced by AngII. In addition, YQFM inhibited cell apoptosis by increasing Bcl-2/Bax ratio and decreasing caspase 3 and p53 expressions in vitro and vivo. Meanwhile, YQFM regulated the autophagy pathway by down-regulating of LC3II and Beclin-1 expressions, as well as up-regulating of p62 expression. Finally, the results showed that YQFM could activate the PI3K/AKT/mTOR pathway by enhancing the p-AKT, p-PI3K and p-mTOR expressions. CONCLUSION: Our results displayed that YQFM attenuated the cardiac hypertrophy by apoptosis inhibition and autophagy regulation via PI3K/AKT/mTOR pathway.

Doxorubicin nanomedicine based on ginsenoside Rg1 with alleviated cardiotoxicity and enhanced antitumor activity.

Aim: The authors aimed to develop Dox@Rg1 nanoparticles with decreased cardiotoxicity to expand their application in cancer. Materials & methods: Dox@Rg1 nanoparticles were developed by encapsulating doxorubicin (Dox) in a self-assembled Rg1. The antitumor effect of the nanoparticles was estimated using 4T1 tumor-bearing mice and the protective effect on the heart was investigated in vitro and in vivo. Results: Different from Dox, the Dox@Rg1 nanoparticles induced increased cytotoxicity to tumor cells, which was decreased in cardiomyocytes by the inhibition of apoptosis. The study in vivo revealed that the Dox@Rg1 nanoparticles presented a perfect tumor-targeting ability and improved antitumor effects. Conclusion: Dox@Rg1 nanoparticles could enhance the antitumor effects and decrease the cardiotoxicity of Dox.

Lay abstract Doxorubicin (Dox) is a drug used to treat cancer; however, it can be toxic to the heart. In this study, researchers made nanoparticles containing Dox and a component of ginseng, a root similar to ginger. They tested the nanoparticles in mice with tumors. The nanoparticles appeared to gather at the tumor site in greater amounts than free Dox. In healthy mice, the nanoparticles gathered less in the heart than free Dox. This means that putting Dox into nanoparticles such as these could improve their anticancer effect and decrease harm to the heart.

Specific protein kinase C isoform exerts chronic inhibition on the slowly activating delayed-rectifier potassium current by affecting channel trafficking.

The slowly activating delayed rectifier K+ current (IKs) plays a key role in the repolarization of ventricular action potential in the human heart and is formed by the pore-forming α-subunit encoded by KCNQ1 (Kv7.1) and ß-subunit encoded by KCNE1. Evidence suggested that IKs was regulated through protein kinase C (PKC) pathway, but the mechanism is controversial. This study was designed to identify the specific PKC isoform involved in the long-term regulation of IKs current. The IKs current was recorded using whole-cell patch-clamp technique in human embryonic kidney (HEK) 293B cell co-transfected with human KCNQ1/KCNE1 genes. The results revealed that both chronic activation of Ang II and PMA reduced the IKs current in a long-term regulation (about 24 hours). Further evidence showed that PKCε knockdown by siRNA antagonized the AngII-induced chronic inhibition on the IKs current, whereas knockdown of cPKC (PKCα and PKCß) attenuated the inhibition effect of PMA on the current. Moreover, the forward transport inhibition of the channel with brefeldin A alleviated the Ang II-induced chronic inhibition on IKs current, while the channel endocytosis inhibition with dynasore alleviated both Ang II and PMA-induced chronic inhibition on IKs current. The above results showed that PKCε activation promoted the channel endocytosis and inhibited the channel forward transport to the plasma membrane, while cPKC activation only promoted the channel endocytosis, which both down regulated the channel current.

The paradox of dopamine and angiotensin II-mediated Na(+), K(+)-ATPase regulation in renal proximal tubules.

Accumulated studies reported that the natruretic dopamine (DA) and the anti-natruretic angiotensin II (Ang II) represent an important mechanism to regulate renal Na(+) and water excretion through intracellular secondary messengers to inhibit or activate renal proximal tubule (PT) Na(+), K(+)-ATPase (NKA). The antagonistic actions were mediated by the phosphorylation of different position of NKA α1-subunit and different Pals-associated tight junction protein (PATJ) PDZ domains, the different protein kinase C (PKC) isoforms (PKC-ß, PKC-ζ), the common adenylyl cyclase (AC) pathway, and the crosstalk and balance between DA and Ang II to NKA regulation. Besides, Ang II-mediated NKA modulation has bi-phasic effects.

Effects of D-Chiro-Inositol on Glucose Metabolism in db/db Mice and the Associated Underlying Mechanisms.

In this study, we observed the effect of D-chiro-inositol (DCI) on glucose consumption in type 2 diabetic db/db mice, and investigated the relevant mechanism. We discovered that the stability of 24-h blood glucose under the nonfasting condition and decreased glucose tolerance were both alleviated after treatment with DCI. Moreover, the content of glycosylated protein and advanced glycation end products in the serum was reduced, the damage in the liver tissue was alleviated, and the synthesis of liver glycogen was significantly promoted. In addition, DCI increased the expression of insulin receptor substrate 2 (IRS2), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), glucose transporters 4 (GLUT4), and phospho-AKT (S473) protein. In contrast, DCI decreased the expression level of glycogen synthase kinase 3ß (GSK3ß) protein in liver tissue to various degrees, as shown by immunohistochemistry and western blotting. Furthermore, DCI increased the mRNA expression of IRS2, PI3K, AKT, and GLUT4, and reduced that of GSK3ß in liver tissue, as demonstrated by polymerase chain reaction. Finally, DCI promoted glucose consumption in high glucose-stimulating HepG2 cells and increased the expression of IRS2 protein in HepG2 cells, as revealed by fluorescence staining and flow cytometry. Our results indicate that DCI can significantly improve glucose metabolism in diabetic mice and HepG2 cells. This effect may be associated with the upregulation of IRS2, PI3K, AKT, and GLUT4 and downregulation of GSK3ß.

Protective effects of rutin on liver injury in type 2 diabetic db/db mice.

The aim of this study was to evaluate the protective effect of rutin on the liver of type 2 diabetic mice and explore the correlation mechanism. The db/db mice, selected as the type 2 diabetes mellitus (T2DM) models, have random blood glucose (RBG) and glucose level after 2 h of oral glucose loading of more than 16.7 mmol/L. After administration of 120 mg/kg or 60 mg/kg rutin, to T2DM mice, RBG, oral glucose tolerance, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, and advanced glycation end products (AGEs) in vivo and vitro of different groups were detected. The liver pathological changes were observed under light and electron microscopy. Western blotting was used to detect the protein expression of insulin receptor substrate 2 (IRS-2) and phosphorylation of phosphatidylinositol 3 kinase (PI3K) on p85, Akt on Ser473, glycogen synthase kinase 3ß (GSK-3ß) on Ser9, real-time quantitative PCR was used to detect IRS-2 mRNA expression. Moreover, dynamically observing the effect of rutin on the generation of AGEs in non-enzymatic protein glycosylated system, Cell Counting Kit-8 (CCK-8) method was used to detect the effect of rutin on proliferation activity of HepG2 liver cells. The results showed that RBG and glucose levels of oral glucose tolerance test (OGTT) of mice in model group were significantly higher than that of normal group, which were significantly reduced after the rutin treatment. Rutin could reduce the ALT, AST activities and AGEs level in serum and potentiate the expression of IRS-2, P-PI3K (p85), P-Akt (Ser473), P-GSK-3ß (Ser9) protein and IRS-2 mRNA in the liver tissue of db/db mice. Moreover, rutin could significantly alleviate the structure disorder of liver, reduce the degeneration and necrosis of liver cells and formation of collagen fibers of db/db mice. The results in vitro also showed that rutin could obviously inhibit the generation of AGEs, and promoted the proliferation activity of high glucose-stimulating HepG2 cells. In general, the protective effect of rutin on the liver of T2DM may be mediated by facilitating signal transduction and activated state of insulin IRS-2/PI3K/Akt/GSK-3ß signal pathway, promoting hepatocyte proliferation, reducing blood glucose level and generation of AGEs.

Imaging of reactive oxygen species burst from mitochondria using laser scanning confocal microscopy.

OBJECTIVE: Although several methods have been used to detect the intracellular reactive oxygen species (ROS) generation, it is still difficult to determine where ROS generate from. This study aimed to demonstrate whether ROS generate from mitochondria during oxidative stress induced mitochondria damage in cardiac H9c2 cells by laser scanning confocal microscopy (LSCM). METHODS: Cardiac H9c2 cells were exposed to H2 O2 (1200µM) to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE); ROS generation was measured by staining cells with dichlorodihydrofluorescein diacetate (H2 DCFDA). RESULTS: A rapid/transient ROS burst from mitochondria was induced in cardiac cells treated with H2 O2 compared with the control group, suggesting that mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells. Meanwhile, the TMRE fluorescence intensity of mitochondria which had produced a great deal of ROS decreased significantly, indicating that the burst of ROS induces the loss of ΔΨm. In addition, the structure of mitochondria was damaged seriously after ROS burst. However, we also demonstrated that the TMRE fluorescence intensity might be affected by H2 DCFDA. CONCLUSIONS: Mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells and these findings provide a new method to observe whether ROS generate from mitochondria by LSCM. However, these observations also suggested that it is inaccurate to test the fluorescence intensities of cells stained with two or more different fluorescent dyes which should be paid more attention to.