Qufeng tongluo decoction decreased proteinuria in diabetic mice by protecting podocytes via promoting autophagy.

Background: Diabetic kidney disease (DKD) is one of diabetic complications, which has become the leading cause of end-stage kidney disease. In addition to angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker(ACEI/ARB) and sodium-glucose cotransporter-2 inhibitor (SGLT2i), traditional Chinese medicine (TCM) is an effective alternative treatment for DKD. In this study, the effect of Qufeng Tongluo (QFTL) decoction in decreasing proteinuria has been observed and its mechanism has been explored based on autophagy regulation in podocyte. Methods: In vivo study, db/db mice were used as diabetes model and db/m mice as blank control. Db/db mice were treated with QFTL decoction, rapamycin, QFTL + 3-Methyladenine (3-MA), trehalose, chloroquine (CQ) and QFTL + CQ. Mice urinary albumin/creatinine (UACR), nephrin and autophagy related proteins (LC3 and p62) in kidney tissue were detected after intervention of 9 weeks. Transcriptomics was operated with the kidney tissue from model group and QFTL group. In vitro study, mouse podocyte clone-5 (MPC-5) cells were stimulated with hyperglycemic media (30 mmol/L glucose) or cultured with normal media. High-glucose-stimulated MPC-5 cells were treated with QFTL freeze-drying powder, rapamycin, CQ, trehalose, QFTL+3-MA and QFTL + CQ. Cytoskeletal actin, nephrin, ATG-5, ATG-7, Beclin-1, cathepsin L and cathepsin B were assessed. mRFP-GFP-LC3 was established by stubRFP-sensGFP-LC3 lentivirus transfection. Results: QFTL decoction decreased the UACR and increased the nephrin level in kidney tissue and high-glucose-stimulated podocytes. Autophagy inhibitors, including 3-MA and chloroquine blocked the effects of QFTL decoction. Further study showed that QFTL decoction increased the LC3 expression and relieved p62 accumulation in podocytes of db/db mice. In high-glucose-stimulated MPC-5 cells, QFTL decoction rescued the inhibited LC3 and promoted the expression of ATG-5, ATG-7, and Beclin-1, while had no effect on the activity of cathepsin L and cathepsin B. Results of transcriptomics also showed that 51 autophagy related genes were regulated by QFTL decoction, including the genes of ATG10, SCOC, ATG4C, AMPK catalytic subunit, PI3K catalytic subunit, ATG3 and DRAM2. Conclusion: QFTL decoction decreased proteinuria and protected podocytes in db/db mice by regulating autophagy.

Erratum to "Phillyrin Mitigates Apoptosis and Oxidative Stress in Hydrogen Peroxide-Treated RPE Cells through Activation of the Nrf2 Signaling Pathway".

[This corrects the article DOI: 10.1155/2020/2684672.].

Paris saponin â induce toxicity in zebrafish by up-regulation of p53 pathway and down-regulation of wnt pathway.

Paris saponin I, II, and VII are three important components in Paris polyphylla, which have been widely studied as tumor cytotoxic drugs, but their safety in vivo has not been reported. Therefore, this study evaluated the safety of these three drugs based on the zebrafish model. Firstly, the lethality curves and lethal concentration of 50% (LC50) values of the three saponins were determined and the results showed the values of LC50 of Paris saponin I, II, and VII were 122.2, 210.7, 566.2 ng/mL, respectively. And then our data revealed that Paris saponin I, II and VII had definite hepatotoxicity, as shown by their significant reduction in the liver area and fluorescence intensity of zebrafish. Besides, Paris saponin Ⅰ affected the heart rate of zebrafish obviously, suggesting its cardiovascular toxicity. Afterwards, we found Paris saponin Ⅰ and Ⅶ reduced the area and fluorescence intensity of kidney in zebrafish, and had mild nephrotoxicity. And when treated with Paris saponin I, the pathological section of liver tissue in zebrafish showed vacuoles, severe necrosis of hepatocytes, and then the apoptosis of hepatocytes could be observed by TUNEL staining. Eventually, we found that the genes expression of p53, Bax and ß-catenin changed significantly in the administration group of Paris saponin I. In general, our study proved Paris saponin Ⅰ was the most toxic of the three saponins, and the most definite toxic target sites were liver and cardiovascular. And it was further inferred that the totoxicity of Paris saponin Ⅰ may be related to the regulation of p53 pathway and Wnt pathway. These results above showed the toxicity of the three saponins in zebrafish, suggesting their safety should be paid more attention in the future.

Isoliquiritigenin attenuates emodin-induced hepatotoxicity in vivo and in vitro through Nrf2 pathway.

Emodin (EMO), the main bioactive component of Polygonum multiflorum, Rheum palmatum, Aloe vera and Cassia acutifolia, can cause severe hepatotoxicity. Isoliquiritigenin (ISL), a flavonoid compound from the Glycyrrhiza, has been reported to be the most potent antioxidant response element (ARE)-luciferase inducer among the main components of licorice. But the protective effect and underlying mechanism of ISL on liver injury induced by EMO has not been reported. This study aims to explore the role of nuclear transcription factor 2 (Nrf2) in EMO-induced hepatotoxicity and the protective effect of ISL. EMO treatment caused cytotoxicity in L-02 cells. Combined treatment of EMO with ISL effectively reversed changes in cell viability, reduced reactive oxygen species (ROS) generation and malondialdehyde (MDA) generation, enhanced the levels of glutathione (GSH) and super oxide dismutase (SOD) induced by EMO in L-02 cells. Furthermore, ISL could also phosphorylate mitogen-activated protein kinases (MAPKs) and up-regulate Kelth-like ECH-associated protein (Keap1). The pathways of MAPKs and Keap1 lead to the separation of Keap1 and Nrf2. Free Nrf2 transferred to the nucleus and enhanced the expression of phase II detoxification enzymes. In conclusion, our results are the first to highlight the beneficial role and relevant mechanisms of ISL in EMO-induced liver injury and provide novel insight into its application.

Shenlian (SL) Decoction, a Traditional Chinese Medicine Compound, May Ameliorate Blood Glucose via Mediating the Gut Microbiota in db/db Mice.

Shenlian (SL) decoction is a herbal formula composed of Coptis and ginseng, of which berberine and ginsenoside are the main constituents. Even though SL decoction is widely used in treating diabetes in China, the mechanism of its antidiabetes function still needs further study. Gut microbiota disorder is one of the important factors that cause diabetes. To explore the effect of SL decoction on intestinal microbiota, gut microbiota of mice was analyzed by sequencing the gut bacterial 16S rRNA V3+V4 region and metagenomics. In this study, results demonstrated that SL decoction had a better hypoglycemic effect and ß cell protection effect than either ginseng or Coptis chinensis. Alpha diversity analysis showed that all interventions with ginseng, Coptis, and SL decoction could reverse the increased diversity and richness of gut microbiota in db/db mice. PCoA analysis showed oral SL decoction significantly alters gut microbiota composition in db/db mice. 395 OTUs showed significant differences after SL treatment, of which 37 OTUs enriched by SL decoction showed a significant negative correlation with FBG, and 204 OTUs decreased by SL decoction showed a significant positive correlation with FBG. Results of KEGG analysis and metagenomic sequencing showed that SL decoction could reduce the Prevotellaceae, Rikenellaceae, and Helicobacteraceae, which were related to lipopolysaccharide biosynthesis, riboflavin metabolism, and peroxisome, respectively. It could also upregulate the abundance of Bacteroidaceae, which contributed to the metabolism of starch and sucrose as well as pentose-glucuronate interconversions. In the species level, SL decoction significantly upregulates the relative abundance of Bacteroides_acidifaciens which showed a significant negative correlation with FBG and was reported to be a potential agent for modulating metabolic disorders such as diabetes and obesity. In conclusion, SL decoction was effective in hypoglycemia and its mechanism may be related to regulating gut microbiota via upregulating Bacteroides_acidifaciens.

Phillyrin Mitigates Apoptosis and Oxidative Stress in Hydrogen Peroxide-Treated RPE Cells through Activation of the Nrf2 Signaling Pathway.

Oxidative stress-induced dysfunction or apoptosis in retinal pigment epithelial (RPE) cells is an important cause of dry age-related macular degeneration (AMD). Although phillyrin has been shown to exert significant antioxidant effects, the underlying mechanism of action remains unclear. The purpose of this study was to investigate the protective effect of phillyrin on hydrogen peroxide- (H2O2-) induced oxidative stress damage in RPE cells and the potential mechanism involved. It was found that phillyrin significantly protected RPE cells from H2O2 cytotoxicity. Furthermore, phillyrin alleviated oxidative stress-induced apoptosis via inhibition of endogenous and exogenous apoptotic pathways. Compared with the H2O2-treated group, the expressions of cleaved caspase-3, cleaved caspase-9, cleaved polymerase (PARP), death receptor Fas, and cleaved caspase-8, as well as Bax/Bcl-2 ratio were decreased in RPE cells after the phillyrin intervention. In addition, phillyrin reversed the oxidative stress-induced reductions in superoxide dismutase (SOD) and glutathione (GSH) levels and annulled the elevations in reactive oxygen species (ROS) and malondialdehyde (MDA), thereby restoring oxidant-antioxidant homeostasis. Phillyrin treatment upregulated the expressions of cyclin E, cyclin-dependent kinase 2 (CDK2), and cyclin A and downregulated the expressions of p21 and p-p53, thereby reversing the G0/G1 cell cycle arrest in H2O2-treated RPE cells. Pretreatment with phillyrin also increased the expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2), total Nrf2, heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductases-1 (NQO-1) in RPE cells and inhibited the formation of Kelch-like ECH-associated protein 1 (Keap1)/Nrf2 protein complex. Thus, phillyrin effectively protected RPE cells from oxidative stress through activation of the Nrf2 signaling pathway and inhibition of the mitochondria-dependent apoptosis pathway.

Itraconazole exerts anti-liver cancer potential through the Wnt, PI3K/AKT/mTOR, and ROS pathways.

Hepatocellular carcinoma (HCC) is one of the most common cancers with the highest morbidity and mortality. It is necessary to develop new anti-liver cancer drugs. Itraconazole is a popular systemic anti-fungal drug with a strong anti-tumor effect. However, so far, it is not clear whether itraconazole has specific anti-tumor effect on liver cancer. The purpose of this study was to investigate itraconazole resistant effect of liver cancer and to explore its potential anti-cancer mechanism. The effect of itraconazole on the proliferation of liver cancer cells was studied with MTT assay. Flow cytometry was used to determine the effect of itraconazole on apoptosis, cell cycle distribution, changes in intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). In addition, after DAPI staining, nuclear morphological changes were observed under the fluorescent microscope, and the release of lactate dehydrogenase (LDH) was measured using the microplate reader. Finally, the expressions of proteins related to the anti-tumor signaling pathway were determined by Western blotting. The results showed that itraconazole significantly inhibited the proliferation of HepG2 and Bel-7405 cells. In addition, the data showed that itraconazole induced apoptosis in HepG2 cells, increased the production of ROS, blocked cell cycle, and decreased MMP. Furthermore, itraconazole inhibited HCC cell growth and promoted apoptosis through the Hh, Wnt/catenin, AKT/mTOR/S6K, ROS and death receptor pathways. Finally, we come to the conclusion that itraconazole exerts anti-liver cancer effect, and has potential for use as a new drug for liver cancer in clinic.

Triptolide Induces Apoptosis Through Fas Death and Mitochondrial Pathways in HepaRG Cell Line.

Triptolide isolated from the traditional Chinese herb Tripterygium wilfordii Hook F., possesses anti-tumor, anti-fertility, and anti-inflammatory properties. Triptolide-induced hepatotoxicity has continued to engage the attention of researchers. However, not much is yet known about the cytotoxicity of triptolide, and the precise mechanisms involved. In the present study, we investigated the cytotoxicity of triptolide and its underlying mechanisms, using the in vitro model (HepaRG cell). The results demonstrated that triptolide significantly reduced cell viability and induced apoptosis in HepaRG cells in a dose- and time-dependent manner. Triptolide treatment also provoked reactive oxygen species (ROS) generation and depolarization of mitochondrial membrane potential (MMP). Moreover, triptolide dose-dependently increased the protein expression levels of Fas, Bax, p53, p21, cyclin E, cleaved caspase-3, 8, and 9; and subsequent cleavage of poly (ADP-ribose) polymerase (PARP). However, the protein expression of Bcl-2, cyclin A, and CDK 2 were significantly decreased. These results suggest that triptolide inhibits cell proliferation and induces apoptosis via the Fas death pathway and the mitochondrial pathway.

Emodin induces apoptosis in human hepatocellular carcinoma HepaRG cells via the mitochondrial caspase­dependent pathway.

Emodin­induced hepatotoxicity in vivo and in vitro has been gaining increasing attention. However, the exact molecular pathways underlying these effects remain poorly clarified. The aim of the present study was to evaluate the cytotoxic effect of emodin on HepaRG cells and to define the underlying mechanism. The results demonstrated that emodin evidently inhibited HepaRG cell growth in a dose­ and time­dependent manner by blocking cell cycle progression in the S and G2/M phase and by inducing apoptosis. Emodin treatment also resulted in generation of reactive oxygen species (ROS), which abrogated mitochondrial membrane potential (MMP). The above effects were all suppressed by antioxidants, such as N­acetylcysteine (NAC). Further studies by western blot analysis howed that emodin upregulated p53, p21, Bax, cyclin E, cleaved caspase­3, 8 and 9, and cleaved poly(ADP­ribose)polymerase (PARP). However, the protein expression of Bcl­2, cyclin A and CDK2 was downregulated. Taken together, our results suggest that emodin induces apoptosis via the mitochondrial apoptosis pathway through cell cycle arrest and ROS generation in HepaRG cells.

Simultaneous Analysis of Quercetin and Naringenin in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry: Application to a Pharmacokinetic Study After Oral Administration.

A rapid and specific LC-MS-MS method has been developed for simultaneous analysis of quercetin and naringenin in rat plasma. The method was applied to the pharmacokinetics studies of quercetin and naringenin after oral administration of Pollen Typhae extract. The samples were prepared by the protein precipitation method. The analysis was carried out on an ACQUITY UPLC™ BEH C18 column with gradient elution using mobile phase, which included acetonitrile and water (containing 0.1% formic acid). The flow rate was 0.4 mL/min. All analytes including internal standard (IS) were monitored by selected reaction monitoring with an electrospray ionization source. Linear responses were obtained for quercetin ranging from 0.5 to 100 ng/mL and naringenin ranging from 5 to 1000 ng/mL. The intra- and interday precisions (RSD) were less than 10.78 and 11.20%. The extraction recovery of the analytes was acceptable. Stability studies showed that quercetin and naringenin were stable in the preparation and analytical process. The validated method was successfully used to determine the concentration-time profiles of quercetin and naringenin.

A novel method to analyze hepatotoxic components in Polygonum multiflorum using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

Polygonum multiflorum, called Heshouwu in China, is a traditional Chinese medicine used to treat various diseases. However, the administration of P. multiflorum (PM) and P. multiflorum Praeparata (PMP) causes numerous adverse effects. This study sought to analyze the toxic components of PM using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), and their hepatotoxicity in L02 human liver cells. Toxicity was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) leakage, and liver enzyme secretion (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) assays. Furthermore, UPLC-Q-TOF/MS, Progenesis QI, and Makerlynx XS software analyses were used to differentiate extracts and analyze the toxic components. The order of toxicity was P. multiflorum ethanol extract (PME)>P. multiflorum water extract (PMW)>P. multiflorum Praeparata ethanol extract (PMPE)>P. multiflorum Praeparata water extract (PMPW), which was determined by MTT assay, LDH leakage, and liver enzyme secretion levels. The analysis methods suggest that PM toxicity may be associated with anthraquinone, emodin-O-(malonyl)-hex, emodin-O-glc, emodin, emodin-8-O-glc, emodin-O-(acetyl)-hex, and emodin-O-hex-sulphate. The toxic mechanisms of these components require further study.

Traditional usages, botany, phytochemistry, pharmacology and toxicology of Polygonum multiflorum Thunb.: a review.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum multiflorum Thunb., which is known as Heshouwu ( in Chinese) in China. It is traditionally valued and reported for hair-blacking, liver and kidney-tonifying and anti-aging effects as well as low toxicity. The aim of this review is to provide comprehensive information on the botany, traditional uses, phytochemistry, pharmacological research and toxicology of Polygonum multiflorum, based on the scientific literature. Moreover, trends and perspectives for future investigation of this plant are discussed. It will build up a new foundation for further study on Polygonum multiflorum. MATERIALS AND METHODS: A systematic review of the literature on Polygonum multiflorum was performed using several resources, including classic books on Chinese herbal medicine and various scientific databases, such as PubMed, SciFinder, the Web of Science, Science Direct, China Knowledge Resource Integrated (CNKI). RESULTS: Polygonum multiflorum is widely distributed throughout the world and has been used as a traditional medicine for centuries in China. The ethnomedical uses of Polygonum multiflorum have been recorded in many provinces of China and Japan for nine species of adulterants in six families. More than 100 chemical compounds have been isolated from this plant, and the major components have been determined to be stilbenes, quinones, flavonoids and others. Crude extracts and pure compounds of this plant are used as effective agents in pre-clinical and clinical practice due to their anti-aging, anti-hyperlipidaemia, anti-cancer and anti-inflammatory effects and to promote immunomodulation, neuroprotection, and the curing of other diseases. However, these extracts can also lead to hepatotoxicity, nephrotoxicity and embryonic toxicity. Pharmacokinetic studies have demonstrated that the main components of Polygonum multiflorum, such as 2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucopyranoside and emodin are distributed among many organs and tissues. CONCLUSION: Therapeutic potential of Polygonum multiflorum has been demonstrated in the conditions like Alzheimer׳s disease, Parkinson׳s disease, hyperlipidaemia, inflammation and cancer, which is attributed to the presence of various stilbenes, quinones, flavonoids, phospholipids and other compounds in the drug. On the other hand, the adverse effects (hepatotoxicity, nephrotoxicity, and embryonic toxicity) of this plant were caused by the quinones, such as emodin and rhein. Thus more pharmacological and toxicological mechanisms on main active compounds are necessary to be explored, especially the combined anthraquinones (Emodin-8-O-ß-d-glucopyranoside, Physcion-8-O-ß-d-glucopyranoside, etc.) and the variety of stilbenes.

Simultaneous determination of 14 constituents of Radix polygoni multiflori from different geographical areas by liquid chromatography-tandem mass spectrometry.

Radix polygoni multiflori (RPM) has antioxidative, anti-aging, liver-protective and antihuman cytomegalovirus activity. It has been proved to be hepatotoxic. Considering multiple ingredients to control RPM quality is essential. The aim of this study was to establish a simple, rapid method using resolution liquid chromatography coupled with a triple quadruple mass spectrometry to identify and quantify the major bioactive constituents in RPM. The method was applied to analyze 14 marker batches from manufacturers from the same province. The ultrasonic extracts of all samples were determined by LC-MS/MS, and assessed by hierarchical cluster analysis. The proposed method was applied to analyze 21 batches of samples with acceptable linearity (R(2) , 0.9930-0.9998), precision (relative standard deviation, RSD, 0.45-4.73%) repeatability (RSD, 1.14-9.41%), stability (RSD, 1.29-12.88%) and recovery (RSD, 1.80-12.15%) of the 14 compounds. Furthermore, the hierarchical cluster analysis was applied to classify 21 samples on the basis of characteristics of the 14 compound markers. The developed method was demonstrated to be simple, sensitive and reproducible, and has significant importance and comprehensive evaluation for quality control of RPM and related preparations. Hierarchical cluster analysis clearly indicated that the RPM from the same province was similar, whereas samples of RPM from different provinces were significantly different.

Simultaneous Determination of Typhaneoside and Isorhamnetin-3-O-Neohesperidoside in Rats After Oral Administration of Pollen Typhae Extract by UPLC-MS/MS.

For the first time, a selective and rapid ultra-performance liquid chromatography method with tandem mass spectrometric (UPLC-MS/MS) detection for simultaneous determination of typhaneoside and isorhamnetin-3-O-neohesperidoside in rat plasma was developed and validated, which was applied to the pharmacokinetic study of Pollen Typhae extract. The separation was carried out on an ACQUITY UPLC(TM) BEH C18 column with gradient elution using mobile phase including acetonitrile and water (containing 0.1% formic acid). The flow rate was 0.4 mL/min. The detection was conducted by means of electrospray ionization mass spectrometry in negative ion mode with multiple reaction monitoring. The assays were linear over the concentration range of 0.5-100 ng/mL, and the lower limit of quantification was 0.5 ng/mL for typhaneoside and isorhamnetin-3-O-neohesperidoside. The method was validated in terms of intra- and interday precision (<9.37%), accuracy (within ±10.91%), linearity, specificity and stability, and has been successfully applied to a pharmacokinetic study of Pollen Typhae extract in rats after oral administration.