Gualou xiebai decoction ameliorates cardiorenal syndrome type II by regulation of PI3K/AKT/NF-κB signalling pathway.

BACKGROUND: Cardiorenal syndromes type II (CRS2) is a multi-organ ailment that manifests as a combination of cardiac and renal dysfunction, resulting in chronic kidney disease due to chronic cardiac insufficiency. It affects at least 26 million people worldwide, and its prevalence is increasing. Gualou Xiebai Decoction (GXD), a traditional Chinese medicine (TCM) with a rich history of application in the management of coronary artery disease, has been explored for its potential therapeutic benefits in CRS2. Nevertheless, the mechanism by which GXD alleviates CRS2 remains obscure, necessitating further investigation. PURPOSE: The aim of this study was to assess the effects of the ethanolic extract of GXD on CRS2 and to elucidate the underlying mechanism in a rat model of myocardial infarction, offering a potential target for clinical treatment for CRS2. STUDY DESIGN AND METHODS: A rat model of CRS2 was induced by surgical myocardial infarction and treated with GXD for 10 weeks. Cardiac function was assessed using echocardiography, while serum and urine biochemistry were analyzed to evaluate potential cardiac and renal damage. Furthermore, tissue samples were obtained for histological, protein, and genetic investigations. In addition, network pharmacology analysis and molecular docking were utilized to predict the primary active compounds, potential therapeutic targets, and interventional pathways through which GXD could potentially exert its effects on CRS2. Subsequently, these predictions were confirmed in vivo and vitro through various analyses. RESULTS: The current investigation employed echocardiography to exhibit the apparent cardiac remodeling following the induction of myocardial infarction. Damage to the heart and kidneys of CRS2 rats was effectively ameliorated by administration of GXD. The outcomes derived from the analyses of HE and Masson staining indicated that the pathological damage to the heart and kidney tissues of rats in the GXD groups was considerably alleviated. Using network pharmacology analysis, AKT1, IL-6, and TNF-α were identified as plausible therapeutic targets for the treatment of CRS with GXD. Subsequent functional and pathway enrichment analysis of the underlying targets disclosed that the PI3K/AKT/NF-κB signaling pathway may be involved in the mechanism of GXD in the treatment of CRS2. Immunohistochemical, western blot, RT-PCR and immunofluorescence staining were employed to demonstrate that GXD can regulate the PI3K/AKT/NF-κB signaling pathway in the CRS2 rat model. Ultimately, administration of the PI3K/AKT agonist 740Y-P counteracted the effect of diosmetin, which was one of the potential active components of GXD analysed by compound-target-disease network, on p-PI3K and p-AKT in vitro. CONCLUSIONS: The findings of this study suggest that GXD improves cardiac and renal function in CRS2 rats and that the underlying mechanism involves inhibition of the PI3K/AKT/NF-κB pathway.

Diosmetin-7-O-ß-D-glucopyranoside suppresses endothelial-mesenchymal transformation through endoplasmic reticulum stress in cardiac fibrosis.

Diosmetin-7-O-ß-D-glucopyranoside (Diosmetin-7-O-glucoside) is a natural flavonoid glycoside known to have a therapeutic application for cardiovascular diseases. Cardiac fibrosis is the main pathological change in the end stage of cardiovascular diseases. Endothelial-mesenchymal transformation (EndMT) induced by endoplasmic reticulum stress (ER stress) via Src pathways is involved in the process of cardiac fibrosis. However, it is unclear whether and how diosmetin-7-O-glucoside regulates EndMT and ER stress to treat cardiac fibrosis. In this study, molecular docking results showed that diosmetin-7-O-glucoside bound well to ER stress and Src pathway markers. Diosmetin-7-O-glucoside suppressed cardiac fibrosis induced by isoprenaline (ISO) and reduced the levels of EndMT, ER stress in mice heart. Primary cardiac microvascular endothelial cells (CMECs) were induced by transforming growth factor-ß1 (TGF-ß1) to perform EndMT. Diosmetin-7-O-glucoside could effectively regulate EndMT and diminish the accumulation of collagen I and collagen III. We also showed that the tube formation in CMECs was restored, and the capacity of migration was partially inhibited. Diosmetin-7-O-glucoside also ameliorated ER stress through the three unfolded protein response branches, as evidenced by organelle structure in transmission electron microscopy images and the expression of protein biomarkers like the glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP). Further analysis showed that diosmetin-7-O-glucoside could suppress the expression level of Src phosphorylation, then block EndMT with the maintenance of endothelial appearance and endothelial marker expression. These results suggested that the diosmetin-7-O-glucoside can regulate EndMT through ER stress, at least in part via Src-dependent pathways.

Toll-like receptor 4 signaling pathway mediates both liver and kidney injuries in mice with hepatorenal syndrome.

Hepatorenal syndrome (HRS) is a complication of cirrhosis with high morbidity and mortality. Nevertheless, the underlying mechanism involving how kidney injury aggravates the progression of cirrhosis remains unclear. This study aims to explore the role of the Toll-like receptor 4 (TLR4) signaling pathway in mediating liver and kidney injuries in HRS mice induced by unilateral ureteral obstruction (UUO) and/or bile duct ligation (BDL). Two weeks after UUO, there were no obvious pathological changes in mouse liver and the unligated side of kidney. Nevertheless, impaired liver and kidney functions, inflammatory response, and fibrosis were examined in mice after 2 wk of BDL. Compared with those of other groups, mice in the BDL + UUO group presented severer liver and kidney injuries, higher levels of inflammatory factors, and faster deposition of collagens, suggesting that kidney injuries accelerated the aggravation of HRS. Correlation analysis identified a positive correlation between expression levels of inflammatory factors and fibrotic levels. Meanwhile, TLR4 and its ligand MyD88 were upregulated during the process of liver and kidney injuries in HRS mice. Further animal experiments in transgenic TLR4-/- mice or in those treated with TAK242, a small molecule inhibitor of TLR4, showed that blocking the TLR4 signaling pathway significantly improved survival quality and survival rate in HRS mice by alleviating liver fibrosis and kidney injury. It is concluded that kidney dysfunction plays an important role in the aggravation of cirrhosis, which may be attributed to the TLR4 signaling pathway. Targeting TLR4 could be a promising therapeutic strategy for protecting both liver and kidneys in patients with HRS.NEW & NOTEWORTHY Our study established BDL, UUO, and BDL + UUO models, providing a novel idea for analyzing liver and kidney diseases. It is highlighted that the kidney injury accelerated the aggravation of HRS via inflammatory response, which could be protected by inhibiting the TLR4 signaling pathway. We believed that targeting TLR4 was a promising therapeutic strategy for protecting both liver and kidney functions in patients with HRS.

Total C-21 Steroidal Glycosides From Baishouwu Ameliorate Hepatic and Renal Fibrosis by Regulating IL-1ß/MyD88 Inflammation Signaling.

Fibrosis is a worldwide public health problem, which typically results from chronic diseases and often leads to organ malfunction. Chronic inflammation has been suggested to be the major trigger for fibrogenesis, yet mechanisms by which inflammatory signals drive fibrogenesis have not been fully elucidated. Total C-21 steroidal glycosides (TCSG) from Baishouwu are the main active components of the root of Cynanchum auriculatum Royle ex Wight, which exert hepatoprotective and anti-inflammation properties. In this study, we established a mouse model with the coexistence of hepatic and renal fibrosis and aimed to investigate the effects of TCSG from Baishouwu on fibrosis and explored the potential mechanisms. The results of biochemical and pathological examinations showed that TCSG from Baishouwu improved liver and kidney function and alleviated hepatic and renal fibrosis by reducing collagen and extracellular matrix deposition in bile duct ligation and unilateral ureteral occlusion (BDL&UUO) mice. According to network pharmacology analysis, the mechanisms underlying the effects of TCSG from Baishouwu on hepatic and renal fibrosis were associated with inflammatory response pathways, including "Signaling by interleukins", "MAP kinase activation", "MyD88 cascade initiated on plasma membrane", and "Interleukin-1 family signaling". Regression analysis and western blot results revealed that IL-1ß/MyD88 inflammation signaling played an essential role in the anti-fibrotic effects of TCSG from Baishouwu. Further data displayed that TCSG from Baishouwu affected inflammatory response and extracellular matrix deposition via suppressing the activation of p38 MAPK/JNK and NF-κB p65 signaling cascades both in the liver and kidney of BDL&UUO mice. Thus, our findings suggest TCSG from Baishouwu as a natural regimen against hepatic and renal fibrosis and provide direct evidence that IL-1ß/MyD88 signaling crucially contributes to hepatic and renal fibrosis and modulates liver-kidney crosstalk by maintaining tight control over inflammatory responses.

[Effect of C21 steroidal glycoside of Cynanchum auriculatum on liver and kidney fibrosis through TLR4 pathway].

The liver and kidney fibrosis model was established by thioacetamide(TAA) and unilateral ureteral obstruction(UUO) in SD rats. The rats were randomly divided into three groups: model group, low and high-dose groups of C21 steroidal glycosides of Cynanchum auriculatum. Another blank control group was set. Four weeks later, serum was taken to detect the biochemical indexes of liver and kidney function. Urine protein and urine creatinine were detected by kits. Liver and kidney tissue samples were stained with HE and Masson staining, and hydroxyproline content was detected. Western blot was used to detect expressions of fibrotic proteins, inflammatory factors and TLR4 signaling pathways, so as to observe the preventive and therapeutic effects of C21 steroidal glycosides from C. auriculatum on hepatic and renal fibrosis and explore its molecular mechanism. Four weeks later, serum biochemical results showed that liver and kidney functions were seriously damaged, and pathological sections showed that inflammatory cell infiltration, decrease of parenchymal cells, and increase of interstitial fibrosis in liver and kidney tissues. The results showed that low and high doses(150, 300 mg·kg~(-1)) of C21 steroidal glycosides could significantly reduce the collagen deposition and the pathological changes of liver and kidney fibrosis compared with the model group. At the same time, we found that the expression levels of TLR4 and MyD88 signaling pathway proteins were significantly increased in the liver and kidney tissues of the model group, and a large number of NF-κB signaling pathway proteins migrated into the nucleus. On the contrary, the expression levels of TLR4, MyD88 signaling pathway proteins and the nuclear migration of NF-κB were significantly inhibited in the low and high dose groups of C21 steroidal glycosides from C. auriculatum. Therefore, it was speculated that the mechanism of C21 steroidal glycoside for preventive and therapeutic effect on hepatic and renal fibrosis was related to inhibit TLR4/MYD88/NF-κB inflammatory pathway, thus preventing hepatic and renal fibrosis.

[Effects of total C-21 steroidal glucosides from Cynanchum auriculatum on oxidative stress pathway in mice with liver injury].

The study aimed to investigate the mechanism of hepatoprotective effect of C-21 steroidal glucosides from Cynanchum auriculatum( Baishouwu) on oxidative stress in mice with liver injury. Mice were randomly divided into normal group,model group,positive control group,Baishouwu high group and Baishouwu low group. The liver injury model was induced by intraperitoneal injection of CCl4 peanut oil solution. All mice were sacrificed to collect blood and liver specimens. The activities of serum levels of ALT and AST were detected. The content of MDA and the activity of SOD in liver homogenate were examined by colorimetry method. Tissues were stained with hematoxylin-eosin for histological examination. The hepatic protein expressions of NF-κB p65,p-IκBα,i NOS and COX-2 were detected by Western blot. The mRNA expressions of TNF-α and IL-6 were determined by RT-PCR. It was found that treatment with C-21 steroidal glucosides from Baishouwu successfully attenuated liver injury induced by CCl4,as shown by decreased levels of serum biochemical indicators( AST,ALT)( P<0. 01). Administration of total C-21 steroidal glucosides enhanced the activity of SOD( P<0. 01) and decreased the content of MDA( P<0. 01) in liver homogenate. Microscopic features suggested that treatment with C-21 steroidal glucosides from Baishouwu was effective in inhibiting CCl4-induced hepatocyte edema and degeneration. Further studies showed that NF-κB p65 overexpression induced by CCl4 was decreased by C-21 steroidal glucosides,leading to the markedly down-regulated protein expression levels of p-IκBα,i NOS and COX-2,as well as the depression of TNF-α and IL-6 mRNA expressions. In conclusion,total C-21 steroidal glucosides from Baishouwu exhibited potent effect on oxidative stress pathway in mice with liver injury induced by CCl4,with enhanced activity of SOD,decreased content of MDA,and down-regulated levels of NF-κB p65,p-IκBα,i NOS and COX-2.

Baishouwu Extract Suppresses the Development of Hepatocellular Carcinoma via TLR4/MyD88/NF-κB Pathway.

Purpose: The root of Cynanchum auriculatum Royle ex Wight, known as Baishouwu, has been widely used for a tonic supplement since ancient times. The current study was performed to explore the effect of Baishouwu extract on the development of experimental hepatocellular carcinoma (HCC) and the potential mechanism involved. Methods: Rats were injected diethylnitrosamine (DEN) to initiate the multistep hepatocarcinogenesis. Animals were treated concurrently with Baishouwu extract given daily by oral gavage for 20 weeks to evaluate its protective effects. Time series sera and organ samples from each group were collected to evaluate the effect of Baishouwu extract on hepatic carcinogenesis. Results: It was found that Baishouwu extract pretreatment successfully attenuated liver injury induced by DEN, as shown by decreased levels of serum biochemical indicators (AST, ALT, ALP, TP, and T-BIL). Administration of Baishouwu extract inhibited the fibrosis-related index in serum and live tissue, respectively from inflammation stage to HCC stage after DEN treatment. It significantly reduced the incidence and multiplicity of DEN-induced HCC development in a dose-dependent manner. Macroscopic and microscopic features suggested that pretreatment with Baishouwu extract for 20 weeks was effective in inhibiting DEN-induced inflammation, liver fibrosis, and HCC. Furthermore, TLR4 overexpression induced by DEN was decreased by Baishouwu extract, leading to the markedly down-regulated levels of MyD88, TRAF6, NF-κB p65, TGF-ß1 and α-SMA in hepatitis, cirrhosis, and hepatocarcinoma. Conclusion: In conclusion, Baishouwu extract exhibited potent effect on the development of HCC by altering TLR4/MyD88/ NF-κB signaling pathway in the sequence of hepatic inflammation-fibrosis-cancer, which provided novel insights into the mechanism of Baishouwu extract as a candidate for the pretreatment of HCC in the future.

Total C-21 steroidal glycosides, isolated from the root tuber of Cynanchum auriculatum Royle ex Wight, attenuate hydrogen peroxide-induced oxidative injury and inflammation in L02 cells.

Oxidative stress plays an important role in the pathology of liver disorders. Total C­21 steroidal glycosides (TCSGs), isolated from the root tuber of Cynanchum auriculatum Royle ex Wight, have been reported to exert numerous effects, including liver protective and antioxidant effects. In order to investigate the potential mechanisms underlying the protective effects of TCSGs on liver function, the present study used the human normal liver cell line, L02, to evaluate the effects of TCSGs on hydrogen peroxide (H2O2)­induced oxidative injury and inflammatory responses. The L02 cells were pretreated with various concentrations of TCSGs, followed by exposure to 1.5 mM H2O2. Cell viability was determined by a 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assay. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and nitric oxide (NO) were measured using colorimetric assays. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH­Px) and the production of malondialdehyde (MDA) were also determined. Intracellular reactive oxygen species (ROS) levels were detected using a fluorescent probe. H2O2­induced oxidative toxicity was attenuated following treatment with TCSGs, as indicated by the increase in cell viability, the decreased levels of ALT, AST, LDH, NO, MDA and ROS, and the increased activities of SOD, CAT and GSH­Px. To further explore the possible mechanisms of action of TCSGs, the nuclear factor erythroid 2­related factor 2 (Nrf2) and nuclear factor­κB (NF)­κB pathways were examined. The results revealed that treatment with TCSGs markedly induced Nrf2 nuclear translocation and upregulated the expression of heme oxygenase­1 (HO­1) in the L02 cells damaged by H2O2. In addition, pretreatment with TCSGs inhibited the NF­κB signaling pathway by blocking the degradation of the inhibitor of nuclear factor κBα (IκBα), thereby reducing the expression and nuclear translocation of NF­κB, as well as reducing the expression of tumor necrosis factor­α (TNF­α), interleukin-6 (IL­6), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX­2). On the whole, the findings of this study demonstrate that TCSGs can protect L02 cells against H2O2­induced oxidative toxicity and inflammatory injury by increasing the expression of Nrf2 and HO­1, mediated by the NF­κB signaling pathway.

[Effects of total C-21 steroidal glycosides from Baishouwu on proliferation invasion and apoptosis of human hepatic cancer HepG2 cells].

This study aimed to investigate the inhibitory effect of total C-21 steroidal glycoside (TCSG) from Baishouwu on the proliferation, invasion and apoptosis of human hepatoma HepG2 cells in vitro and the relevant molecular mechanism. The experiment was divded into control group, TCSG groups (25, 60, 150 mg·L⁻¹) and positive control cisplatin group (1.33 mg·L⁻¹). Human hepatocyte L-02 cells and hepatoma HepG2 cells were treated with different concentrations of TCSG. Then, the inhibitory effect of TCSG on the proliferation of HepG2 cells was detected by CCK-8 method. Cell cycle, cell apoptosis and mitochondrial membrane potential were detected by flow cytometry. The apoptotic morphology was observed by Hoechst 33258 staining. Cell migration and invasion abilities were analyzed by Transwell chamber model. The protein expressions of Bcl-2, Bax, caspase 3, cleaved caspase 3 and Cyt C (cytosolchondrial) were detected by Western blot. Compared with the control group, the proliferation of HepG2 cells was significantly inhibited after treatment with different concentrations of TCSG for 48 h in a dose-dependent manner(P<0.01), but no obvious effect was observed on the proliferation of L-02 cells. After treatment with TCSG for 48 h, apoptotic morphology such as nuclear shrinkage, fragmentation and semilunar or circular was observed; migration and invasion abilities of cells were significantly decreased, cell cycle was blocked in the G0/G1 phase(P<0.01), mitochondrial membrane potential was remarkably decreased(P<0.01), and so did the ratio of apoptosis(P<0.01).Western blot results showed that the protein expressions of Bax, caspase 3, cleaved caspase 3, and Cyt C were significantly up-regulated(P<0.05, P<0.01), while the Bcl-2 protein was significantly down-regulated(P<0.05, P<0.01). Furthermore, the ratio of Bax/Bcl-2 was increased (P<0.01). The results suggested that TCSG could inhibit the proliferation and invasion of HepG2 cells, and induce the apoptosis of HepG2 cells. The potential mechanism may be related to the blocking of cell cycle and the regulation of the expressions of apoptosis-related proteins by activating mitochondrial pathway.

Nanoemulsion delivery system of tea polyphenols enhanced the bioavailability of catechins in rats.

Tea polyphenols (TP) were emulsified with corn oil and polysorbate 80 by high-pressure homogenization. The oil in water (O/W) TP nanoemulsion had droplet sizes of 99.42±1.25nm after preparation. The TP nanoemulsion was stable during storage at 4, 25 or 40°C for 20days. An in vitro simulated digestion assay showed that the bioaccessibility of (-)-epigallocatechin gallate (EGCG) was increased in the nanoemulsion compared to that in aqueous solution, but that the bioaccessibilities of (-)-epigallocatechin (EGC), (-)-epicatechin (EC) and (-)-gallocatechin gallate (GCG) were greatly decreased. Compared with rats fed an aqueous TP solution, rats fed the TP nanoemulsion had significantly lower maximum plasma concentrations (Cmax) of EGCG and EGC, but the area under the plasma concentration-time curve (AUC0-t) was increased. The data show that use of a nanoemulsion system to deliver tea polyphenols may enhance the absorption of EGCG through controlled release.

Salidroside, A Natural Antioxidant, Improves ß-Cell Survival and Function via Activating AMPK Pathway.

Aim: The enhanced oxidative stress contributes to progression of type 2 diabetes mellitus (T2DM) and induces ß-cell failure. Salidroside is a natural antioxidant extracted from medicinal food plant Rhodiola rosea. This study was aimed to evaluate protective effects of salidroside on ß-cells against diabetes associated oxidative stress. Methods and Results: In diabetic db/db and high-fat diet-induced mice, we found salidroside ameliorated hyperglycemia and relieved oxidative stress. More importantly, salidroside increased ß-cell mass and ß-cell replication of diabetic mice. Mechanism study in Min6 cells revealed that, under diabetic stimuli, salidroside suppressed reactive oxygen species production and restore mitochondrial membrane potential (ΔΨm) via reducing NOX2 expression and inhibiting JNK-caspase 3 apoptotic cascade subsequently to protect ß-cell survival. Simultaneously, diabetes associated oxidative stress also activated FOXO1 and triggered nuclear exclusion of PDX1 which resulted in ß-cell dysfunction. This deleterious result was reversed by salidroside by activating AMPK-AKT to inhibit FOXO1 and recover PDX1 nuclear localization. The efficacy of salidroside in improving ß-cell survival and function was further confirmed in isolated cultured mouse islets. Moreover, the protective effects of salidroside on ß-cells against diabetic stimuli can be abolished by an AMPK inhibitor compound C, which indicated functions of salidroside on ß-cells were AMPK activation dependent. Conclusion: These results confirmed beneficial metabolic effects of salidroside and identified a novel role for salidroside in preventing ß-cell failure via AMPK activation. Our finding highlights the potential value of Rhodiola rosea as a dietary supplement for diabetes control.

Antitumor evaluation and multiple analysis on different extracted fractions of the root of Cynanchum auriculatum Royle ex Wight.

The root of Cynanchum auriculatum (C. auriculatum) Royle ex Wight has been shown to possess various pharmacological effects and has recently attracted much attention with respect to its potential role in antitumor activity. The C-21 steroidal glycosides are commonly accepted as the major active ingredients of C. auriculatum. In this study, the antitumor abilities of different extracted fractions of the root bark and the root tuber of C. auriculatum were investigated by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in human cancer cell lines HepG2 and SMMC-7721. The results showed that the chloroform and ethyl acetate fractions of the root tuber suppressed tumor cell growth strongly. To identify and characterize the chemical constituents of different active fractions, an ultra high performance liquid chromatography with triple-quadrupole tandem mass spectrometry method was developed for the simultaneous quantitation of eight C-21 steroidal glycosides. The analysis revealed that the C-21 steroidal glycosides were concentrated in the chloroform and ethyl acetate fractions, and the total contents of different fractions in the root tuber were significantly higher than those of corresponding ones in the root bark. Furthermore, the C-21 steroidal glycosides based on different types of aglucones were prone in different medicinal parts of C. auriculatum.

Metabolic profiling of the hepatotoxicity and nephrotoxicity of Ginkgolic acids in rats using ultra-performance liquid chromatography-high-definition mass spectrometry.

Ginkgolic acids (GAs) are thought to be the potentially hazardous constituents corresponding to the toxic side effects of Ginkgo products. In this study, toxicological and metabolomics studies of GAs were carried out by ultra-performance liquid chromatography-high-definition mass spectrometry (UPLC-HDMS). Significant changes in serum clinical chemistry were observed in the both low (100 mg/kg) and high (900 mg/kg) doses. Especially the serum enzyme of ALT, AST, LDH, and CK decreased in treated groups. The histopathological observation demonstrated hepatic steatosis in liver and tubular vacuolar degeneration in kidney. These results demonstrated the hepatotoxicity and nephrotoxicity of GAs. Functional disorders are more likely to be toxic induced by GAs. Metabolic profiling within seven days revealed the change of the body status after oral administration. The results indicated the body function was significantly influenced at the 3rd day and could recover in seven days. Metabolomic analysis showed alterations in 14 metabolites from plasma such as LysoPC(18:0), LysoPC(18:2) and other lipids. The results suggested that exposure to GAs could cause disturbances in liver and kidney function associated with the metabolisms of lipids, glucose and the enzyme activity.

Hepatic inflammation-fibrosis-cancer axis in the rat hepatocellular carcinoma induced by diethylnitrosamine.

PURPOSE: Hepatocellular carcinoma (HCC) cases are closely associated with chronic inflammation and fibrosis which is known as hepatic inflammation-fibrosis-cancer (IFC) axis. The aim of this study is to elucidate the development characteristics of the rat HCC model based on IFC axis. METHODS: The diethylnitrosamine (DEN)-induced rat HCC, which presents a stepwise histopathological progression that is similar to human HCC, was used to analyze the features of the different stages (inflammation, fibrosis, cancer). Rats were injected DEN at a dose of 30 mg/kg body weight twice a week for 11 weeks and the animals were observed until week 20. Time series sera and organ samples from the DEN animal model were collected to evaluate the dynamic changes. RESULTS: It was found that serum biochemical indicators (AST, ALT, ALP, TP, T-BIL, IL-6, TNF-α) from DEN-treated group were higher than that from control group. Fibrosis-related index in serum and live tissue were increased, respectively, from week 4 after DEN treatment. The expression of TGF-ß1 and α-SMA in DEN-treated group was higher than that in control group. JAK2/STAT3 signaling was significantly up-regulated in DEN-treated group compared to that in control group. The histological examination confirmed that the hepatocarcinogenesis model was successfully established, and 100% of the animals in the DEN-exposed group developed liver tumors at 20 weeks. According to the pathological changes, the model characterized resulted in three stages: the inflammation stage (week 2-6), the fibrosis stage (week 8-12), and the HCC stage (week 14-20). CONCLUSIONS: The results suggested that the HCC development was associated with IFC axis. The serial progression of hepatocarcinogenesis was according to the sequence of hepatic inflammation, fibrosis and then hepatic tumor.

Characterization of Collagen Peptides in Elaphuri Davidiani Cornu Aqueous Extract with Proliferative Activity on Osteoblasts Using Nano-Liquid Chromatography in Tandem with Orbitrap Mass Spectrometry.

First documented in Shennong Bencao Jing (about 200 B.C.-200 A.D.), Elaphuri Davidiani Cornu (EDC) has been recorded for its effects in strengthening bones and balancing other aspects of overall health for approximately 2000 years. In the present study, our aim was to investigate which are the components of the active EDC fraction by a peptidomic strategy. We explored the extent to which EDC increases the proliferation of osteoblasts by measuring the elevations in osteonectin and type I collagen mRNA levels and characterized it using nano-flow liquid chromatography in tandem with orbitrap mass spectrometry. In total, 272 peptide sequences from collagens were determined. "Hot regions" in parent proteins determined by peptide heat maps which indicated that amino acid sequences in the regions might undergo proteolysis easily and generate peptides. Among the identified peptides, 90.2% were hydrophilic, and the molecular weight of 97.1% of identified peptides was lower than 2000 Da. According to these results, EDC collagen-derived peptides were easily analyzed and identified. Moreover, this methodology is feasible to characterize the active peptides matrices originated from collagen hydrolysates or some other animal horn- derived TCMs.

Gualou Xiebai decoction inhibits cardiac dysfunction and inflammation in cardiac fibrosis rats.

BACKGROUND: Gualou Xiebai Decoction (GXD) is a well-known traditional Chinese recipe. It has been used to treat cardiovascular disorders for nearly two thousand years. But there is a lack of reports on cardiac fibrosis and underlying mechanism. METHODS: Myocardial infarction was performed by ligation of left anterior descending coronary artery (LAD) in male Wistar rats. Rats with myocardial infarction were treated with GXD (1.14 g/kg, 4.53 g/kg) daily for 4 weeks. Cardiac function was evaluated by echocardiography. Hemodynamic parameters and infarct size were measured in each group. Myocardial enzymes were examined by biochemical tests. Inflammatory cytokines were assessed by ELISA, and interrelated proteins were detected by western blot. RESULTS: Cardiac function was significantly improved in GXD-treatment rats after myocardial infarction (MI), which was accompanied with decreased infarct size. Administration of GXD to myocardial fibrosis rats significantly ameliorated the activities of AST, LDH and CK-MB in serum. The increase in inflammatory factors (TNF-α, IL-1ß) were markedly reduced upon GXD treatment. Furthermore, the inflammatory mediators (NF-κB p65, TNF-α, MCP-1) were down-regulated by GXD in the myocardial fibrosis rats. CONCLUSIONS: Treatment with GXD improved cardiac function induced by myocardial fibrosis by inhibiting expression of inflammatory mediators associated with NF-κB.

Two New Oleanane-Type Saponins with Anti-Proliferative Activity from Camellia oleifera Abel. Seed Cake.

Two new oleanane-type saponins, named oleiferasaponins C4 (1) and C5 (2), were isolated from Camellia oleifera Abel. seed cake residue. Their respective structures were identified as 16α-hydroxy-22α-O-angeloyl-23α-aldehyde-28-dihydroxymethylene-olean-12-ene-3ß-O-[ß-d-galacto-pyranosyl-(1→2)]-[ß-d-glucopyranosyl-(1→2)-ß-d-galactopyranosy-(1→3)]-ß-d-glucopyranosid-uronic acid methyl ester (1) and 16α-hydroxy-22α-O-angeloyl-23α-aldehyde-28-dihydroxy-methylene-olean-12-ene-3ß-O-[ß-d-galactopyranosyl-(1→2)]-[ß-d-galactopyranosyl-(1→3)]-ß-d-glucopyranosiduronic acid methyl ester (2) through 1D- and 2D-NMR, HR-ESI-MS, and GC-MS spectroscopic methods. The two compounds exhibited potent cytotoxic activities against five human tumor cell lines (BEL-7402, BGC-823, MCF-7, HL-60 and KB).

Puerarin Protects Pancreatic ß-Cells in Obese Diabetic Mice via Activation of GLP-1R Signaling.

Diabetes is characterized by a loss and dysfunction of the ß-cell. Glucagon-like peptide 1 receptor (GLP-1R) signaling plays an important role in ß-cell survival and function. It is meaningful to identify promising agents from natural products which might activate GLP-1R signaling. In this study, puerarin, a diet isoflavone, was evaluated its beneficial effects on ß-cell survival and GLP-1R pathway. We showed that puerarin reduced the body weight gain, normalized blood glucose, and improved glucose tolerance in high-fat diet-induced and db/db diabetic mice. Most importantly, increased ß-cell mass and ß-cell proliferation but decreased ß-cell apoptosis were observed in puerarin-treated diabetic mice as examined by immunostaining of mice pancreatic sections. The protective effect of puerarin on ß-cell survival was confirmed in isolated mouse islets treated with high glucose. Further mechanism studies showed that the circulating level of GLP-1 in mice was unaffected by puerarin. However, puerarin enhanced GLP-1R signaling by up-regulating expressions of GLP-1R and pancreatic and duodenal homeobox 1, which subsequently led to protein kinase B (Akt) activation but forkhead box O1 inactivation, and promoted ß-cell survival. The protective effect of puerarin was remarkably suppressed by Exendin(9-39), an antagonist of GLP-1R. Our study demonstrated puerarin improved glucose homeostasis in obese diabetic mice and identified a novel role of puerarin in protecting ß-cell survival by mechanisms involving activation of GLP-1R signaling and downstream targets.

Effects of Ziziphus jujuba fruit extracts on cytochrome P450 (CYP1A2) activity in rats.

Drug-drug interactions have become a serious problem in the clinic, since plant-based medicines are extensively used. The present study investigated the effects of Ziziphus jujuba fruit (ZJ) extract on the pharmacokinetics of phenacetin, a typical substrate of a cytochrome P450 enzyme CYP 1A2, in rats. The rats were pretreated with the water extract (1.0 g · kg(-1)) or the ethanolic extract (3.6 g · kg(-1)) of ZJ for 10 days, and the pharmacokinetics of phenacetin was investigated after intravenous administration. In an in vitro assay, acetaminophen formation in the hepatic microsomes of ZJ-treated rats was investigated to assess CYP1A2 activity. Our results demonstrated that the treatment with the water and ethanolic extracts of ZJ decreased the plasma concentration of phenacetin and increased the plasma concentration of acetaminophen, resulting in a 43.2% and 15.5% reduction in the AUC0-120 of phenacetin, respectively, and a 53.2% and 64.9% increase in the AUC0-120 of acetaminophen, respectively after intravenous administration. The water or ethanolic extract of ZJ significantly increased the clearance of phenacetin and acetaminophen formation in hepatic microsomes. In conclusion, ZJ extracts displayed effects on the pharmacokinetics of phenacetin and increased the CYP1A2 activity in rats. Therefore, precaution on drug-drug interactions should be taken when ZJ is co-administered with drugs metabolized by CYP1A2, which may result in decreased concentrations of these drugs.