Geniposide alleviates heart failure with preserved ejection fraction in mice by regulating cardiac oxidative stress via MMP2/SIRT1/GSK3ß pathway.

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome with cardiac dysfunction, fluid retention and reduced exercise tolerance as the main manifestations. Current treatment of HFpEF is using combined medications of related comorbidities, there is an urgent need for a modest drug to treat HFpEF. Geniposide (GE), an iridoid glycoside extracted from Gardenia Jasminoides, has shown significant efficacy in the treatment of cardiovascular, digestive and central nervous system disorders. In this study we investigated the therapeutic effects of GE on HFpEF experimental models in vivo and in vitro. HFpEF was induced in mice by feeding with HFD and L-NAME (0.5 g/L) in drinking water for 8 weeks, meanwhile the mice were treated with GE (25, 50 mg/kg) every other day. Cardiac echocardiography and exhaustive exercise were performed, blood pressure was measured at the end of treatment, and heart tissue specimens were collected after the mice were euthanized. We showed that GE administration significantly ameliorated cardiac oxidative stress, inflammation, apoptosis, fibrosis and metabolic disturbances in the hearts of HFpEF mice. We demonstrated that GE promoted the transcriptional activation of Nrf2 by targeting MMP2 to affect upstream SIRT1 and downstream GSK3ß, which in turn alleviated the oxidative stress in the hearts of HFpEF mice. In H9c2 cells and HL-1 cells, we showed that treatment with GE (1 µM) significantly alleviated H2O2-induced oxidative stress through the MMP2/SIRT1/GSK3ß pathway. In summary, GE regulates cardiac oxidative stress via MMP2/SIRT1/GSK3ß pathway and reduces cardiac inflammation, apoptosis, fibrosis and metabolic disorders as well as cardiac dysfunction in HFpEF. GE exerts anti-oxidative stress properties by binding to MMP2, inhibiting ROS generation in HFpEF through the SIRT1/Nrf2 signaling pathway. In addition, GE can also affect the inhibition of the downstream MMP2 target GSK3ß, thereby suppressing the inflammatory and apoptotic responses in HFpEF. Taken together, GE alleviates oxidative stress/apoptosis/fibrosis and metabolic disorders as well as HFpEF through the MMP2/SIRT1/GSK3ß signaling pathway.

Protein Z and Protein Z-dependent protease inhibitor in patients with acute ischemic stroke: A prospective mechanistic study.

OBJECTIVES: Protein Z (PZ) /Protein Z-dependent protease inhibitor (ZPI) (PZ/ZPI) system is a new anticoagulant system discovered in recent years, which plays an important role in many diseases. We aimed to compare the plasma PZ/ZPI levels of acute ischemic stroke (AIS) patients and non-stroke control participants and the role of PZ/ZPI in the development of stroke was preliminarily analyzed. MATERIALS AND METHODS: Enzyme linked immunosorbent assay (ELISA) was used to detect and compare plasma PZ levels of 86 patients with acute AIS and 85 non-stroke control patients. Multivariable Logistic regression was used to analyze whether PZ was an independent risk factor for AIS. RESULTS: In the present study, plasma PZ is closely related to inflammatory response, coagulation process and platelet activation, and may participate in the development of AIS by inducing inflammatory responses and interfering with the coagulation process. CONCLUSIONS: Our results suggested that plasma PZ level is one of the independent risk factors of AIS, and plasma ZPI was closely related to coagulation and platelet parameter and may play a role in the coagulation process during AIS.

6-Methyl flavone inhibits Nogo-B expression and improves high fructose diet-induced liver injury in mice.

Excessive fructose consumption increases hepatic de novo lipogenesis, resulting in cellular stress, inflammation and liver injury. Nogo-B is a resident protein of the endoplasmic reticulum that regulates its structure and function. Hepatic Nogo-B is a key protein in glycolipid metabolism, and inhibition of Nogo-B has protective effects against metabolic syndrome, thus small molecules that inhibit Nogo-B have therapeutic benefits for glycolipid metabolism disorders. In this study we tested 14 flavones/isoflavones in hepatocytes using dual luciferase reporter system based on the Nogo-B transcriptional response system, and found that 6-methyl flavone (6-MF) exerted the strongest inhibition on Nogo-B expression in hepatocytes with an IC50 value of 15.85 µM. Administration of 6-MF (50 mg· kg-1 ·d-1, i.g. for 3 weeks) significantly improved insulin resistance along with ameliorated liver injury and hypertriglyceridemia in high fructose diet-fed mice. In HepG2 cells cultured in a media containing an FA-fructose mixture, 6-MF (15 µM) significantly inhibited lipid synthesis, oxidative stress and inflammatory responses. Furthermore, we revealed that 6-MF inhibited Nogo-B/ChREBP-mediated fatty acid synthesis and reduced lipid accumulation in hepatocytes by restoring cellular autophagy and promoting fatty acid oxidation via the AMPKα-mTOR pathway. Thus, 6-MF may serve as a potential Nogo-B inhibitor to treat metabolic syndrome caused by glycolipid metabolism dysregulation.

Novel flavonoid 1,3,4-oxadiazole derivatives ameliorate MPTP-induced Parkinson's disease via Nrf2/NF-κB signaling pathway.

Parkinson's disease (PD) is a progressive neurodegenerative disorder with a complex etiology. Neuroinflammation and oxidative stress are important factors driving the progression of PD. It has been reported that 1,3,4-oxadiazole and flavone derivatives have numerous biological functions, especially in the aspect of anti-inflammatory and antioxidant. Based on the strategy of pharmacodynamic combination, we introduced 1,3,4-oxadiazole moiety into the flavonoid backbone, designed and synthesized a series of novel flavonoid 1,3,4-oxadiazole derivatives. Further, we evaluated their toxicity, anti-inflammatory and antioxidant activities using BV2 microglia. Following a comprehensive analysis, compound F12 showed the best pharmacological activity. In vivo, we induced the classical PD animal model by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into C57/BL6J mice. Our results showed that compound F12 ameliorated MPTP-induced dysfunction in mice. Further, compound F12 reduced oxidative stress by promoting the nucleation of nuclear factor erythroid 2-related factor 2 (Nrf2) and decreased the inflammatory response by inhibiting the nuclear translocation of nuclear factor-κB (NF-κB) in vivo and in vitro. Meanwhile, compound F12 inhibited the mitochondrial apoptotic pathway to rescue microglia inflammation-mediated loss of dopaminergic neurons. In conclusion, compound F12 reduced oxidative stress and inflammation and could be as a potential agent for PD treatment.

Fisetin treatment alleviates kidney injury in mice with diabetes-exacerbated atherosclerosis through inhibiting CD36/fibrosis pathway.

Diabetes-related vascular complications include diabetic cardiovascular diseases (CVD), diabetic nephropathy (DN) and diabetic retinopathy, etc. DN can promote the process of end-stage renal disease. On the other hand, atherosclerosis accelerates kidney damage. It is really an urge to explore the mechanisms of diabetes-exacerbated atherosclerosis as well as new agents for treatment of diabetes-exacerbated atherosclerosis and the complications. In this study we investigated the therapeutic effects of fisetin, a natural flavonoid from fruits and vegetables, on kidney injury caused by streptozotocin (STZ)-induced diabetic atherosclerosis in low density lipoprotein receptor deficient (LDLR-/-) mice. Diabetes was induced in LDLR-/- mice by injecting STZ, and the mice were fed high-fat diet (HFD) containing fisetin for 12 weeks. We found that fisetin treatment effectively attenuated diabetes-exacerbated atherosclerosis. Furthermore, we showed that fisetin treatment significantly ameliorated atherosclerosis-enhanced diabetic kidney injury, evidenced by regulating uric acid, urea and creatinine levels in urine and serum, and ameliorating morphological damages and fibrosis in the kidney. In addition, we found that the improvement of glomerular function by fisetin was mediated by reducing the production of reactive oxygen species (ROS), advanced glycosylation end products (AGEs) and inflammatory cytokines. Furthermore, fisetin treatment reduced accumulation of extracellular matrix (ECM) in the kidney by inhibiting the expression of vascular endothelial growth factor A (VEGFA), fibronectin and collagens, while enhancing matrix metalloproteinases 2 (MMP2) and MMP9, which was mainly mediated by inactivating transforming growth factor ß (TGFß)/SMAD family member 2/3 (Smad2/3) pathways. In both in vivo and in vitro experiments, we demonstrated that the therapeutic effects of fisetin on kidney fibrosis resulted from inhibiting CD36 expression. In conclusion, our results suggest that fisetin is a promising natural agent for the treatment of renal injury caused by diabetes and atherosclerosis. We reveal that fisetin is an inhibitor of CD36 for reducing the progression of kidney fibrosis, and fisetin-regulated CD36 may be a therapeutic target for the treatment of renal fibrosis.

Design, synthesis, and SAR study of novel flavone 1,2,4-oxadiazole derivatives with anti-inflammatory activities for the treatment of Parkinson's disease.

Inflammation is one of a major feature of Parkinson's disease (PD) which poses a threat to people's health in the world. It has been reported that antioxidation and anti-inflammation have significant effects on the treatment of PD. 1,2,4-oxadiazole and flavone derivatives have remarkable antioxidant and anti-inflammatory activities. In order to find highly effective drugs for PD treatment, based on the remarkable anti-inflammatory and antioxidant activities of the 1,2,4-oxadiazole pharmacophore and the flavonoid pharmacophore, we designed and synthesized a novel series of 3-methyl-8-(3-methyl-1,2,4-oxadiazol-5-yl)-2-phenyl-4H-chromen-4-one derivatives by pharmacophore combination, and evaluated their anti-inflammatory and antioxidation activities for PD treatment. Preliminary structure-activity relationship (SAR) analysis was conducted by their inhibitory activities against reactive oxygen species (ROS) and NO release in LPS-induced BV2 Microglia cells, and the optimal compound Flo8 exhibited the most potent anti-inflammatory and antioxidant activities. Both in vivo and in vitro results showed that Flo8 inhibited neuronal apoptosis by inhibiting inflammatory and apoptotic signaling pathways. In vivo studies also showed that the compound Flo8 ameliorated motor and behavioral deficits and increased serum dopamine levels in MPTP-induced PD model mice. Taken together, this study demonstrated the compound Flo8 could be a promising agent for the treatment of PD.

Transcription factor 21 accelerates vascular calcification in mice by activating the IL-6/STAT3 signaling pathway and the interplay between VSMCs and ECs.

Vascular calcification is caused by the deposition of calcium salts in the intimal or tunica media layer of the aorta, which increases the risk of cardiovascular events and all-cause mortality. However, the mechanisms underlying vascular calcification are not fully clarified. Recently it has been shown that transcription factor 21 (TCF21) is highly expressed in human and mouse atherosclerotic plaques. In this study we investigated the role of TCF21 in vascular calcification and the underlying mechanisms. In carotid artery atherosclerotic plaques collected from 6 patients, we found that TCF21 expression was upregulated in calcific areas. We further demonstrated TCF21 expression was increased in an in vitro vascular smooth muscle cell (VSMC) osteogenesis model. TCF21 overexpression promoted osteogenic differentiation of VSMC, whereas TCF21 knockdown in VSMC attenuated the calcification. Similar results were observed in ex vivo mouse thoracic aorta rings. Previous reports showed that TCF21 bound to myocardin (MYOCD) to inhibit the transcriptional activity of serum response factor (SRF)-MYOCD complex. We found that SRF overexpression significantly attenuated TCF21-induced VSMC and aortic ring calcification. Overexpression of SRF, but not MYOCD, reversed TCF21-inhibited expression of contractile genes SMA and SM22. More importantly, under high inorganic phosphate (3 mM) condition, SRF overexpression reduced TCF21-induced expression of calcification-related genes (BMP2 and RUNX2) as well as vascular calcification. Moreover, TCF21 overexpression enhanced IL-6 expression and downstream STAT3 activation to facilitate vascular calcification. Both LPS and STAT3 could induce TCF21 expression, suggesting that the inflammation and TCF21 might form a positive feedback loop to amplify the activation of IL-6/STAT3 signaling pathway. On the other hand, TCF21 induced production of inflammatory cytokines IL-1ß and IL-6 in endothelial cells (ECs) to promote VSMC osteogenesis. In EC-specific TCF21 knockout (TCF21ECKO) mice, VD3 and nicotine-induced vascular calcification was significantly reduced. Our results suggest that TCF21 aggravates vascular calcification by activating IL-6/STAT3 signaling and interplay between VSMC and EC, which provides new insights into the pathogenesis of vascular calcification. TCF21 enhances vascular calcification by activating the IL-6-STAT3 signaling pathway. TCF21 inhibition may be a new potential therapeutic strategy for the prevention and treatment of vascular calcification.

Liver X receptor agonists exert antitumor effects against hepatocellular carcinoma via inducing REPS2 expression.

Recent studies show that liver X receptor (LXR) agonists exert significant antitumor effects in a variety of tumor cell lines including hepatocellular carcinoma (HCC). But the molecular mechanisms underlying LXR antitumor activity are not fully understood. In this study we investigated the effect of LXR agonist T0901317 (T317) on HCC development and its relationship with RalA binding protein 1 (RALBP1)-associated EPS domain containing 2 (REPS2)/epidermal growth factor receptor (EGFR) signaling axis. We showed that T317 (0.1-0.5 µM) dose-dependently increased REPS2 expression in normal hepatocytes (BNLCL.2 and LO2) and HCC cells (HepG2 and Huh-7). Using promoter activity assay and chromatin immunoprecipitation (CHIP) assay we demonstrated that T317 enhanced REPS2 expression at the transcriptional level via promoting the binding of LXR protein to the LXR-response element (LXRE) in the REPS2 promoter region. We showed that the inhibitory effect of T317 on the proliferation and migration of HCC cells was closely related to REPS2. Moreover, we revealed that T317 (400 nM) increased expression of REPS2 in HepG2 cells, thus inhibiting epidermal growth factor (EGF)-mediated endocytosis of EGFR as well as the downstream activation of AKT/NF-κB, p38MAPK, and ERK1/2 signaling pathways. Clinical data analysis revealed that REPS2 expression levels were inversely correlated with the development of HCC and reduced REPS2 expression associated with poor prognosis, suggesting that REPS2 might be involved in the development of HCC. In conclusion, this study provides new insights into the potential mechanisms of LXR agonist-inhibited HCC.

Roxadustat alleviates nitroglycerin-induced migraine in mice by regulating HIF-1α/NF-κB/inflammation pathway.

Sensitization of central pain and inflammatory pathways play essential roles in migraine, a primary neurobiological headache disorder. Since hypoxia-inducible factor-1α (HIF-1α) is implicated in neuroprotection and inflammation inhibition, herein we investigated the role of HIF-1α in migraine. A chronic migraine model was established in mice by repeated injection of nitroglycerin (10 mg/kg, i.p.) every other day for 5 total injections. In the prevention and acute experiments, roxadustat, a HIF-1α stabilizer, was orally administered starting before or after nitroglycerin injection, respectively. Pressure application measurement, and tail flick and light-aversive behaviour tests were performed to determine the pressure pain threshold, thermal nociceptive sensitivity and migraine-related light sensitivity. At the end of experiments, mouse serum samples and brain tissues were collected for analyses. We showed that roxadustat administration significantly attenuated nitroglycerin-induced basal hypersensitivity and acute hyperalgesia by improving central sensitization. Roxadustat administration also decreased inflammatory cytokine levels in serum and trigeminal nucleus caudalis (TNC) through NF-κB pathway. Consistent with the in vivo results showing that roxadustat inhibited microglia activation, roxadustat (2, 10, and 20 µM) dose-dependently reduced ROS generation and inflammation in LPS-stimulated BV-2 cells, a mouse microglia cell line, by inhibiting HIF-1α/NF-κB pathway. Taken together, this study demonstrates that roxadustat administration ameliorates migraine-like behaviours and inhibits central pain sensitization in nitroglycerin-injected mice, which is mainly mediated by HIF-1α/NF-κB/inflammation pathway, suggesting the potential of HIF-1α activators as therapeutics for migraine.

A CE-LIF method based on long wavelength fluorescence labeling for the analysis of thiols in human urine.

A rapid and robust CE method using a long wavelength fluorescent reagent 1,7-dimethyl-3,5-distyryl-8-phenyl-(2-maleimide)difluoroboradiaza-s-indacene as the labeling reagent has been developed for the simultaneous determination of thiols, including glutathione, cysteine, homocysteine, N-acetylcysteine, cysteinylglycine, and penicillamine. The derivatization reaction was carried out in 14 mmol/L pH 8.5 borate buffer at 30°C for 6 min and the labeled thiols derivatives were separated with the running buffer containing 30 mmol/L pH 7.4 phosphate, 30% v/v acetonitrile and 8 mmol/L SDS within 12 min. Detection limits ranged from 0.4 to 2.4 nmol/L. To demonstrate the capability of this method, it was applied to the analysis of thiols in human urine with recoveries of 92.4-105.6%. The derivatization reaction was much faster at milder conditions, and the analysis was rapider. Moreover, with excitation wavelength at long wavelength region, background interference from samples was reduced effectively. The present method seems to be a potential choice for quantifying thiols in human urine.

Polycarbonate microspheres containing mitomycin C and magnetic powders as potential hepatic carcinoma therapeutics.

The polycarbonate copolymer poly(trimethylene carbonate-co-5,5-dimethyl trimethylene carbonate) (P(TMC-co-DTC)) was synthesized by the polymerization of trimethylene carbonate (TMC) and 5,5-dimethyl trimethylene carbonate (DTC) using tin (II) 2-ethylhexanoate [Sn(Oct)(2)] as a catalyst. In vitro degradation tests indicated this polycarbonate copolymer degraded slowly in phosphate buffer saline solution (PBS, 0.1 mol/L, at 37°C). Magnetic polymer microspheres (MMC-PC-M) generated from the P(TMC-co-DTC) copolymer and containing Fe(3)O(4) magnetic ultrafine powders and an anticancer drug, mitomycin C (MMC) were prepared by a solvent evaporation technique. These anticancer magnetic polycarbonate microspheres showed strong magnetic responsiveness and high MMC loading capacity. In vitro drug release studies indicated that these microspheres sustained steady release rates of MMC in PBS. In vitro cytotoxicity assays demonstrated the microspheres were strongly inhibitory to human hepatic carcinoma (Bel-7204) cells. In vivo site-specific therapy in nude mice with human hepatic carcinoma indicated that the microspheres possessed markedly high antitumor activity against human hepatic carcinoma (Bel-7204).