Effects of total flavones of Abelmoschus manihot (L.) on the treatment of diabetic nephropathy via the activation of solute carriers in renal tubular epithelial cells.

As a traditional Chinese medicine, Huangkui capsule (HKC) has been used to treat patients with kidney diseases, including diabetic nephropathy (DN). We have recently demonstrated that HKC could re-regulate the activities of solute carriers (SLC)s in proximal and distal convoluted tubules of kidneys in regression of the development of DN. The main active chemical constituents of HKC are the flavones of Abelmoschus manihot (L.). The current study aims to further evaluate the efficacy of total flavones of A. manihot (TFA) in the regression of DN by analyzing SLC activities in proximal and distal convoluted tubules of kidneys. TFA (0.076 g/kg/d) or vehicle was administered in db/db mice, the animal model of type 2 diabetes and DN, daily via oral gavage for four weeks. Blood glucose levels and urinary albumin-to-creatinine ratio (UACR) were measured and used for the determination of T2D and DN. Ten SLCs, including slc2a2, slc4A1, slc5a2, slc5A3, slc5a8, slc6a20, slc27a2, slc12a3, slc34a1 and slc38a2 were highly expressed in proximal and distinct convoluted tubules of kidneys. Their expression at mRNA and protein levels before and after TFA treatment were analyzed with real-time RT-PCR and immunohistochemistry. Data showed that UACR in the db/db mice after TFA treatment was significantly decreased. Compared with the group of non-diabetic control, slc2a2, slc4A1, slc5a2, slc5A3, slc5a8, slc6a20, slc27a2, slc12a3, slc34a1 and slc38a2 in the group of DN were down-regulated but up-regulated after TFA treatment. Further analyses of whole kidney sections indicated that the numbers and structures of the nephron in db/db mice was increased and improved after TFA treatment. Thereby, the current study provides further evidence that the flavones in A. manihot have pharmacological effects on the treatment of DN by improving the biological function of SLCs in kidneys.

Evaluation of the efficacy of Abelmoschus manihot (L.) on diabetic nephropathy by analyzing biomarkers in the glomeruli and proximal and distal convoluted tubules of the kidneys.

Introduction: As a traditional Chinese medicine, Abelmoschus manihot (L.) in the form of Huangkui (HK) capsule has been used as a medication for kidney diseases, including diabetic nephropathy (DN), in China. The most significant effect of HK capsule treatment in kidney diseases is the reduction of albuminuria and proteinuria. To evaluate the efficacy of HK capsule in the regression of DN, in the current study, we analyzed the biomarkers in the glomerulus and proximal and distal convoluted tubules in the kidneys of db/db mice, the animal model for type 2 diabetes and DN. Methods: Huangkui capsules (0.84 g/kg/d) or vehicle were administered daily via oral gavage for 4 weeks in db/db mice. Urinary albumin-to-creatinine ratio and blood glucose levels were measured during the whole experimental period. Five biomarkers in the glomerulus and proximal and distal convoluted tubules in the kidneys were selected, namely, col4a3, slc5a2, slc34a1, slc12a3, and slc4a1, and their activities at mRNA and protein levels before and after HK capsule treatment were analyzed by real-time RT-PCR and immunohistochemistry. Result and discussion: After HK capsule treatment for 4 weeks, the urinary albumin-to-creatinine ratio in db/db mice was found to be significantly decreased. The activities of col4a3, slc5a2, slc34a1, slc12a3, and slc4a1 in the kidneys were upregulated in db/db mice prior to the treatment but downregulated after HK capsule treatment. Further analyses of the fields of whole kidney tissue sections demonstrated that the number of nephrons in the kidneys of db/db mice with HK capsule treatment was higher than that in the kidneys of db/db mice without HK capsule treatment. Thereby, the current study provides experimental evidence confirming the medical efficacy of A. manihot in the reduction of albuminuria and proteinuria, suggesting that A. manihot may have pharmacological efficacy in the regression of the development of type 2 diabetes-DN.

Abelmoschus Manihot ameliorates the levels of circulating metabolites in diabetic nephropathy by modulating gut microbiota in non-obese diabetes mice.

Huangkui capsule (HKC), a traditional Chinese medicine, has been used for medication of kidney diseases, including diabetic nephropathy (DN). The current study aimed to evaluate the effects of HKC in the modulation of gut microbiota and the amelioration of metabolite levels by using non-obese diabetes (NOD) mice with DN. The microbiota from three parts of intestines (duodenum, ileum and colon) in NOD mice with and without HKC treatment were analysed using 16S rDNA sequencing techniques. Untargeted metabolomics in plasma of NOD mice were analysed with liquid mass spectrometry. Results showed that HKC administration ameliorated DN in NOD mice and the flora in duodenum were more sensitive to HKC intervention, while the flora in colon had more effects on metabolism. The bacterial genera such as Faecalitalea and Muribaculum significantly increased and negatively correlated with most of the altered metabolites after HKC treatment, while Phyllobacterium, Weissella and Akkermansia showed an opposite trend. The plasma metabolites, mainly including amino acids and fatty acids such as methionine sulfoxide, BCAAs and cis-7-Hexadecenoic acid, exhibited a distinct return to normal after HKC treatment. The current study thereby provides experimental evidence suggesting that HKC may modulate gut microbiota and subsequently ameliorate the metabolite levels in DN.

Evaluation of Cardiovascular Toxicity of Folic Acid and 6S-5-Methyltetrahydrofolate-Calcium in Early Embryonic Development.

Folic acid (FA) is a synthetic and highly stable version of folate, while 6S-5-methyltetrahydrofolate is the predominant form of dietary folate in circulation and is used as a crystalline form of calcium salt (MTHF-Ca). The current study aims to evaluate the toxicity and safety of FA and MTHF-Ca on embryonic development, with a focus on cardiovascular defects. We began to analyze the toxicity of FA and MTHF-Ca in zebrafish from four to seventy-two hours postfertilization and assessed the efficacy of FA and MTHF-Ca in a zebrafish angiogenesis model. We then analyzed the differently expressed genes in in vitro fertilized murine blastocysts cultured with FA and MTHF-Ca. By using gene-expression profiling, we identified a novel gene in mice that encodes an essential eukaryotic translation initiation factor (Eif1ad7). We further applied the morpholino-mediated gene-knockdown approach to explore whether the FA inhibition of this gene (eif1axb in zebrafish) caused cardiac development disorders, which we confirmed with qRT-PCR. We found that FA, but not MTHF-Ca, could inhibit angiogenesis in zebrafish and result in abnormal cardiovascular development, leading to embryonic death owing to the downregulation of eif1axb. MTHF-Ca, however, had no such cardiotoxicity, unlike FA. The current study thereby provides experimental evidence that FA, rather than MTHF-Ca, has cardiovascular toxicity in early embryonic development and suggests that excessive supplementation of FA in perinatal women may be related to the potential risk of cardiovascular disorders, such as congenital heart disease.

Evaluation of the association between maternal folic acid supplementation and the risk of congenital heart disease: a systematic review and meta-analysis.

BACKGROUND: Folic acid (FA), as a synthetic form of folate, has been widely used for dietary supplementation in pregnant women. The preventive effect of FA supplementation on the occurrence and recurrence of fetal neural tube defects (NTD) has been confirmed. Incidence of congenital heart diseases (CHD), however, has been parallelly increasing worldwide. The present study aimed to evaluate whether FA supplementation is associated with a decreased risk of CHD. METHODS: We searched the literature using PubMed, Web of Science and Google Scholar, for the peer-reviewed studies which reported CHD and FA and followed with a meta-analysis. The study-specific relative risks were used as summary statistics for the association between maternal FA supplementation and CHD risk. Cochran's Q and I2 statistics were used to test for the heterogeneity. RESULTS: Maternal FA supplementation was found to be associated with a decreased risk of CHD (OR = 0.82, 95% CI: 0.72-0.94). However, the heterogeneity of the association was high (P < 0.001, I2 = 92.7%). FA supplementation within 1 month before and after pregnancy correlated positively with CHD (OR 1.10, 95%CI 0.99-1.23), and high-dose FA intake is positively associated with atrial septal defect (OR 1.23, 95%CI 0.64-2.34). Pregnant women with irrational FA use may be at increased risk for CHD. CONCLUSIONS: Data from the present study indicate that the heterogeneity of the association between maternal FA supplementation and CHD is high and suggest that the real relationship between maternal FA supplementation and CHD may need to be further investigated with well-designed clinical studies and biological experiments.

Chemical constituents, clinical efficacy and molecular mechanisms of the ethanol extract of Abelmoschus manihot flowers in treatment of kidney diseases.

Abelmoschus manihot, also called as "Huangkui" in Chinese, is an annual flowering herb plant in the family of Malvaceae. As a traditional Chinese medicine, the ethanol extract of the flower in Abelmoschus manihot is made as Huangkui capsule and has been used for medication of the patients with kidney diseases. Its efficacy in clinical symptoms is mainly improving renal function and reducing proteinuria among the patients with chronic kidney disease, diabetic kidney disease or IgA nephropathy. The possible mechanism of Huangkui capsule treatment in kidney diseases may include reducing inflammation and anti-oxidative stress, improving immune response, protecting renal tubular epithelial cells, ameliorating podocyte apoptosis, glomerulosclerosis and mesangial proliferation, as well as inhibiting renal fibrosis. In this review, we first described chemical constituents and pharmacokinetic characteristics in ethanol extract of the flower of Abelmoschus manihot. We then summarized the clinical and epidemiological relevancies of kidney diseases particularly in the mainland of China and discussed the possible molecular mechanisms of Huangkui capsule in the treatment of kidney diseases. Finally, we prospected further research on cellular and molecular mechanisms and application of this Chinese natural medicine in kidney diseases.

Hypothalamic BMP9 suppresses glucose production by central PI3K/Akt/mTOR pathway.

Bone morphogenetic proteins (BMPs) are secreted ligands that belong to the transforming growth factor-ß (TGF-ß) superfamily. BMP7 has been reported to play a role in reversing obesity and regulating appetite in the hypothalamus. Whether BMP9 plays a central role in regulating glucose metabolism and insulin sensitivity remains unclear. Here, we investigated the impact of central BMP9 signaling and possible route of transmission. We performed intracerebroventricular (ICV) surgery and injected adenovirus expressing BMP9 (Ad-BMP9) into the cerebral ventricle of mice. Metabolic analysis, hyperinsulinemic-euglycemic clamp test, and analysis of phosphatidylinositol 3,4,5-trisphosphate (PIP3) formation were then performed. Real-time PCR and Western blotting were performed to detect gene expression and potential pathways involved. We found that hypothalamic BMP9 expression was downregulated in obese and insulin-resistant mice. Overexpression of BMP9 in the mediobasal hypothalamus reduced food intake, body weight, and blood glucose level, and elevated the energy expenditure in high-fat diet (HFD)-fed mice. Importantly, central treatment with BMP9 improved hepatic insulin resistance (IR) and inhibited hepatic glucose production in HFD-fed mice. ICV BMP9-induced increase in hepatic insulin sensitivity and related metabolic effects were blocked by ICV injection of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) signaling. In addition, ICV BMP9 promoted the ability of insulin to activate the insulin receptor/phosphoinositide 3-kinase (PI3K)/Akt pathway in the hypothalamus. Thus, this study provides insights into the potential mechanism by which central BMP9 ameliorates hepatic glucose metabolism and IR via activating the mTOR/PI3K/Akt pathway in the hypothalamus.

Protective effect of berberine on high glucose and hypoxia-induced apoptosis via the modulation of HIF-1α in renal tubular epithelial cells.

Berberine (BBR), derived from Huanglian (Coptis chinensis), is a traditional Chinese herbal medicine. In the current study, we investigated the effects of BBR in high glucose (HG) and hypoxia-induced apoptosis with normal rat renal tubular epithelial (NRK-52E) and human kidney proximal tubular cells (HK-2) and further explored the underlying molecular mechanism of hypoxia-inducible factor 1α (HIF-1α) in diabetic kidney disease (DKD). Apoptosis in NRK-52E and HK-2 cells induced by HG (30 mM)/hypoxia and anti-apoptosis with BBR pretreatment (30 µM) were analyzed by using the terminal uridine nick 3' end labeling method. Activities of apoptotic proteins and anti-apoptotic factor at mRNA and protein levels were determined with real-time RT-PCR and Western blot. HIF-1α action in the apoptosis with BBR pretreatment or siRNA interfere was investigated with flow-cytometry and Western blot. Up-regulation of apoptotic proteins (Bax cytochrome C, caspase 9 and caspase 3) and down-regulation of anti-apoptotic factor Bcl-xL were accompanied with HG/Hypoxia-induced apoptosis in NRK-52E and HK-2 cells but all reversals were found after BBR pretreatment. Activity of HIF-1α was induced under HG/Hypoxia conditions and up-regulated with BBR pretreatment. Furthermore, knockdown of HIF-1α via siRNA significantly removed the anti-apoptosis effects of BBR, while the BBR-mediated HIF-1α activity was suppressed by the pharmacological inhibition of Akt. The present study thereby provided evidence that BBR protected renal tubular epithelial cells from hypoxia/HG-induced apoptosis through activation of HIF-1α in the PI3K/Akt signal pathway and suggested that BBR could be a potential drug in DKD.

Evaluation of Antidiabetic Effects of the Traditional Medicinal Plant Gynostemma pentaphyllum and the Possible Mechanisms of Insulin Release.

Aims. To evaluate the antidiabetic effects of Gynostemma pentaphyllum (GP) in Goto-Kakizaki (GK) rat, an animal model of type 2 diabetes, and to investigate the mechanisms of insulin release. Methods. Oral glucose tolerance test was performed and plasma insulin levels were measured. Results. An oral treatment with GP (0.3 g/kg of body weight daily) for two weeks in GK rats improved glucose tolerance versus placebo group (P < 0.01). Plasma insulin levels were significantly increased in the GP-treated group. The insulin release from GP-treated GK rats was 1.9-fold higher as compared to the control group (P < 0.001). GP stimulated insulin release in isolated GK rat islets at high glucose. Opening of ATP-sensitive potassium (K-ATP) channels by diazoxide and inhibition of calcium channels by nifedipine significantly decreased insulin response to GP. Furthermore, the protein kinase A (PKA) inhibitor H89 decreased the insulin response to GP (P < 0.05). In addition, GP-induced insulin secretion was decreased after preincubation of GK islets with pertussis toxin to inhibit exocytotic Ge proteins (P < 0.05). Conclusion. The antidiabetic effect of GP is associated with the stimulation of insulin release from the islets. GP-induced insulin release is partly mediated via K-ATP and L-type Ca(2+) channels, the PKA system and also dependent on pertussis toxin sensitive Ge-protein.

Antidiabetic Effect of Oral Borapetol B Compound, Isolated from the Plant Tinospora crispa, by Stimulating Insulin Release.

Aims. To evaluate the antidiabetic properties of borapetol B known as compound 1 (C1) isolated from Tinospora crispa in normoglycemic control Wistar (W) and spontaneously type 2 diabetic Goto-Kakizaki (GK) rats. Methods. The effect of C1 on blood glucose and plasma insulin was assessed by an oral glucose tolerance test. The effect of C1 on insulin secretion was assessed by batch incubation and perifusion experiments using isolated pancreatic islets. Results. An acute oral administration of C1 improved blood glucose levels in treated versus placebo groups with areas under glucose curves 0-120 min being 72 ± 17 versus 344 ± 10 mmol/L (P < 0.001) and 492 ± 63 versus 862 ± 55 mmol/L (P < 0.01) in W and GK rats, respectively. Plasma insulin levels were increased by 2-fold in treated W and GK rats versus placebo group at 30 min (P < 0.05). C1 dose-dependently increased insulin secretion from W and GK isolated islets at 3.3 mM and 16.7 mM glucose. The perifusions of isolated islets indicated that C1 did not cause leakage of insulin by damaging islet beta cells (P < 0.001). Conclusion. This study provides evidence that borapetol B (C1) has antidiabetic properties mainly due to its stimulation of insulin release.

Labisia pumila extract down-regulates hydroxysteroid (11-beta) dehydrogenase 1 expression and corticosterone levels in ovariectomized rats.

We evaluated the effects of a standardized Labisia pumila var. alata (LPva) extract on body weight change, hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) expressions and corticosterone (CORT) level in ovariectomized (OVX) rats. The decoction of LPva has been used for generations among Malay women in Malaysia to maintain a healthy reproductive system.Thirty-six Sprague-Dawley OVX rats were treated orally with LPva extract (10, 20 or 50 mg/kg/day) or estrogen replacement (ERT) for 30 days. Sham operated rats were used as controls. Compared to untreated OVX rats, LPva-treated rats showed less weight gain and had significantly down-regulated HSD11B1 mRNA in liver tissues. HSD11B1 mRNA in adipose tissues increased by 55% (p < 0.05) in OVX rats but normalized in rats treated with LPva. Similarly, there was significant down-regulation (p < 0.05) of protein levels of HSD11B1 in both liver and adipose tissue of LPva and ERT groups, and CORT levels were significantly reduced in both groups of rats. This is the first study ever conducted to evaluate the beneficial effects of LPva in relation to weight gain caused by estrogen insufficiency. Results implied that the bioactive components in LPva extract affect not only HSD11B1 expressions in both adipose and liver tissues but also decrease circulating CORT. The extract should be explored for its potential use as a natural remedy for weight management.

Labisia pumila extract regulates body weight and adipokines in ovariectomized rats.

OBJECTIVE: We investigated the effects of a standardized water extract of Labisia pumila var. alata (LPva), and compared to estrogen replacement (ERT), on body weight gain, uterus weight, adipose tissue mRNA and protein levels of adipokines in ovariectomized (OVX) rats. METHODS: Eight-week-old OVX Sprague-Dawley rats were administered orally with either 10 mg/kg/day (LPva10), 20 mg/kg/day (LPva20) or 50 mg/kg/day (LPva50) of LPva for 30 days. Sham-operated (Sham) and estrogen-treated OVX rats (ERT, 0.625 mg/kg/day) served as controls. Plasma adipokines were measured, and mRNA expressions of the adipokines were determined in the adipose tissues. RESULTS: ERT- and LPva50-treated OVX rats showed significantly less (p<0.05) weight gain compared to untreated OVX rats. Ovariectomy caused plasma leptin levels to decrease significantly (p<0.05), but when treated with LPva or ERT, plasma leptin increased significantly to levels higher or comparable to that seen in the Sham group. The mRNA expression of leptin was higher in the LPva-treated animals than in all other groups. In contrast, the elevated plasma resistin concentrations in OVX rats were significantly reduced in rats given ERT (p<0.05) and LPva extracts (p<0.05). There was no difference in adiponectin levels in all groups. The uterus to body weight ratio of untreated OVX rats was significantly low compared to Sham (p<0.05), but showed dose-dependent increase upon treatment with LPva. CONCLUSION: The present study provides first evidence that LPva exerts uterotrophic effect and regulates body weight gain by modulating secretion of leptin and resistin, and expression of the adipokines in adipose tissues.