Qingshan Li

Apoptosis induced neurotoxicity of Di-n-butyl-di-(4-chlorobenzohydroxamato) Tin (IV) via mitochondria-mediated pathway in PC12 cells.

The severe toxicity of antitumor organotin (IV) compounds limits their application in clinic, however, the toxic mechanism is still unclear. Di-n-butyl-di-(4-chlorobenzohydroxamato) Tin (IV) (DBDCT), an antitumor agent with high activity and obvious neurotoxicity was chosen as a typical diorganotin (IV) compound to investigate its neurotoxic mechanism using PC12 cells and comprehensive methods. Treatment with DBDCT resulted in a dose- and time-dependent growth inhibition of PC12 cells. The changes in cell morphology were observed using light microscopy, fluorescence microscopy and transmission electron microscopy. PC12 cell apoptosis induced by DBDCT was confirmed by annexin V/propidium iodide staining, and characterized by cleavage of caspase-9 and caspase-3 proteins. DBDCT induced the release of cytochrome c from the mitochondria to the cytosol and the generation of reactive oxygen species. DBDCT up-regulated the expression of Bax, down-regulated the expression of Bcl-2, and significantly increased the ratio of Bax/Bcl-2. DBDCT also caused the phosphorylation of JNK and p38(MAPK). In rats exposed to DBDCT, apoptosis was also observed in brain, as shown by the detection of cleaved caspase-9 and caspase-3 proteins and increased TUNEL positive staining. In conclusion, the results demonstrated that DBDCT caused the neurotoxicity by inducing apoptosis via mitochondria-mediated pathway.

Farrerol regulates occludin expression in hydrogen peroxide-induced EA.hy926 cells by modulating ERK1/2 activity.

Endothelial tight junction is a crucial intracellular junctional structure that controls paracellular permeability across vascular endothelium. Oxidative stress-mediated elevation in endothelial permeability is associated with pathogenesis of several cardiovascular diseases. In the present research, the regulation of farrerol on occludin, a transmembrane proteins associated with endothelial tight junction, was investigated in hydrogen peroxide-induced human endothelium-derived EA.hy926 cells. Western blot analysis demonstrated that H2O2 exposure caused a significant decrease in occludin expression, but had little effect on ZO-1 expression, and the decrease of occludin expression was significantly attenuated by farrerol in a dose-dependent manner. Meanwhile, immunofluorescent staining assay also demonstrated that the loss of occludin expression induced by H2O2 exposure was restored by farrerol pretreatment. Further investigations showed that farrerol prevented H2O2-induced activation of extracellular signal-regulated kinase (ERK) 1/2 in a dose-dependent manner. The use of U0126, a specific inhibitor of MEK1/2, proved that H2O2-induced decrease of occludin in EA.hy926 cells was likely associated with activation of ERK1/2, which indicated that the regulation of farrerol on occludin expression in H2O2-induced EA.hy926 cells was likely related to the modulation of ERK1/2 activation. In conclusion, the present study demonstrates for the first time that farrerol has potential effects on oxidative stress-induced endothelial tight junction disruption and suggests that farrerol is a potential candidate for the intervention of endothelial permeability-associated cardiovascular diseases.

Synthesis and biological activity of 4-substituted benzoxazolone derivatives as a new class of sEH inhibitors with high anti-inflammatory activity in vivo.

A series of novel 4-substituted benzoxazolone derivatives was synthesized, characterized and evaluated as human soluble epoxide hydrolase (sEH) inhibitors and anti-inflammatory agents. Some compounds showed moderate sEH inhibitory activities in vitro, and two novel compounds, 3g and 4j, exhibited the highest activities with IC50 values of 1.72 and 1.07 μM, respectively. Structure-activity relationships (SARs) revealed that introduction of a lipophilic amino acid resulted in an obvious increase in the sEH inhibitory activity, especially for derivatives containing a phenyl (3d, IC(50) = 2.67 μM), pyrrolidine (3g, IC(50) = 1.72 μM), or sulfhydryl group (3e, IC(50)=3.02 μM). Several compounds (3a-3g) were tested in vivo using a xylene-induced ear edema mouse model. Three compounds (3d, 3f, and 3g) showed strong anti-inflammatory activities in vivo which were higher than that of Chlorzoxazone, a reference drug widely used in the clinic. Our investigation provided a novel type of sEH inhibitor and anti-inflammatory agent that may lead to the discovery of a potential candidate for clinical use.

Relaxation of Rat Aorta by Farrerol Correlates with Potency to Reduce Intracellular Calcium of VSMCs

Farrerol, isolated from Rhododendron dauricum L., has been proven to be an important multifunctional physiologically active component, but its vasoactive mechanism is not clear. The present study was performed to observe the vasoactive effects of farrerol on rat aorta and to investigate the possible underlying mechanisms. Isolated aortic rings of rat were mounted in an organ bath system and the myogenic effects stimulated by farrerol were studied. Intracellular Ca2+ ([Ca2+]in) was measured by molecular probe fluo-4-AM and the activities of L-type voltage-gated Ca2+ channels (LVGC) were studied with whole-cell patch clamp in cultured vascular smooth muscle cells (VSMCs). The results showed that farrerol significantly induced dose-dependent relaxation on aortic rings, while this vasorelaxation was not affected by NG-nitro-l-arginine methylester ester or endothelium denudation. In endothelium-denuded aortas, farrerol also reduced Ca2+-induced contraction on the basis of the stable contraction induced by KCl or phenylephrine (PE) in Ca2+-free solution. Moreover, after incubation with verapamil, farrerol can induce relaxation in endothelium-denuded aortas precontracted by PE, and this effect can be enhanced by ruthenium red, but not by heparin. With laser scanning confocal microscopy method, the farrerol-induced decline of [Ca2+]in in cultured VSMCs was observed. Furthermore, we found that farrerol could suppress Ca2+ influx via LVGC by patch clamp technology. These findings suggested that farrerol can regulate the vascular tension and could be developed as a practicable vasorelaxation drug.

Chitosan attenuates dibutyltin-induced apoptosis in PC12 cells through inhibition of the mitochondria-dependent pathway.

Dibutyltin (DBT) which was widely used as biocide and plastic stabilizer has been described as a potent neurotoxicant. Chitosan (CS), a natural nontoxic biopolymer, possesses a variety of biological activities including antibacterial, antifungal, free radical scavenging and neuroprotective activities. The present study was undertaken to investigate the protective effects of CS against DBT-induced apoptosis in rat pheochromocytoma (PC12) cells and the underlying mechanisms in vitro. Our results demonstrated that pretreatment with CS significantly increased the cell viability and decreased lactate dehydrogenase (LDH) release induced by DBT in a dose-dependent manner. Meanwhile, DBT-induced cell apoptosis, mitochondrial membrane potential (MMP) disruption, and generation of intracellular reactive oxygen species (ROS) were attenuated by CS. Real-time PCR assay showed that DBT markedly enhanced the mRNA levels of Bax, Bad, cytochrome-c and Apaf-1, reduced the Bcl-2 and Bcl-xL mRNA levels, while these genes expression alteration could be partially reversed by CS treatment. Furthermore, CS also inhibited the DBT-inducted activation of caspase-9, and -3 at mRNA and protein expression levels. Taken together, these results suggested that CS could protect the PC12 cells from apoptosis induced by DBT through inhibition of the mitochondria-dependent pathway.

Apoptosis induced by farrerol in human gastric cancer Sgc-7901 cells through the mitochondrial-mediated pathway

Farrerol, a typical flavanone isolated from the Chinese medicinal plant Rhododendron dauricum L., has been found to show various biological activities. However, to the best of our knowledge, its inhibitory actions against cancer cells have not been reported as yet. Therefore, the present study aimed to investigate the cytotoxic and apoptotic effects of farrerol on human gastric cancer SGC-7901 cells. Farrerol showed a 50% inhibition of SGC-7901 cell growth at a concentration of 40.4 &mgr;mol/l for 24 h according to MTT assays. The cell morphology results indicated that SGC-7901 cells treated with farrerol showed several features of apoptotic cell death, which was also confirmed by the Annexin-V FITC/PI double-staining assay. Further studies showed that farrerol treatment induced the attenuation of mitochondrial membrane potential, accompanied by the release of Cyt-c and the activation of caspase-9 and caspase-3. Furthermore, farrerol decreased the gene expression of Bcl-2, whereas the gene expression level of Bax was found to increase after farrerol treatment. These combined results indicated that farrerol can induce apoptosis through a mitochondrial-mediated pathway.

Oxidative stress in di-n-butyl-di-(4-chlorobenzohydroxamato)tin (IV)-induced hepatotoxicity determined by proteomic profiles

Significant attention has been paid to the antitumor diorganotin(IV) compounds during the last few decades. However, severe toxicity limits their application and the toxic mechanism is still unclear. Of these toxicities, liver is the most important target organ. In this study, di-n-butyl-di-(4-chlorobenzohydroxamato)tin(IV) (DBDCT), an antitumor agent with high activity and obvious hepatotoxicity was chosen as a typical diorganotin(IV) compound to investigate the hepatotoxic mechanism using proteomics methods for the first time. The cell growth, cell morphology, proteomics, ROS, MDA, and GSH were assessed in this study. The results showed that cell growth was inhibited and cell morphology was changed after DBDCT treatment. A total of nine significantly and consistently altered proteins associated with oxidative stress were identified. Among the altered proteins, Trx1 and protein DJ1, that could regulate the oxidative stress process, were chosen for a detailed analysis. They were demonstrated to be up-regulated following exposure to DBDCT at both protein and mRNA levels in a dose- and time-dependant manner, and the consequences were concordant with the experimental results of ROS, MDA and GSH. These findings showed that oxidative stress played a key role in DBDCT-mediated toxicity, and Trx1 may be a potential biomarker for the early diagnosis of hepatotoxicity.

Sustained ERK activation-mediated proliferation inhibition of farrerol on human gastric carcinoma cell line by G0/G1-phase cell-cycle arrest.

Current cancer treatment is partly limited by chemotherapy-induced vascular toxicity associated with damage to vascular endothelial cells. In this study, the cytotoxicity of farrerol against SGC7901 gastric cancer cells and human umbilical vein endothelial cells (HUVECs) in vitro was investigated along with the underlying mechanisms of its growth-inhibitory effect against SGC7901 cells. MTT assays showed that farrerol inhibited SGC7901 cell growth, but exerted no cytotoxicity against HUVECs. Flow cytometry showed that treatment of SGC7901 cells with farrerol (5, 40, or 160 &mgr;mol/l) for 24 h caused G0/G1 cell cycle arrest in a concentration-dependent manner. Western blotting indicated that exposure of SGC7901 cells to farrerol resulted in significant upregulation of p27KIP1 (p27), accompanied by sustained activation of ERK1/2 and p38 MAPK instead of JNK. Farrerol-stimulated p27 expression, p38 MAPK activation, and cell growth inhibition were attenuated by pretreatment with U0126, an MEK1/2 inhibitor. In conclusion, this study indicates the selective cytotoxicity of farrerol against SGC7901 cells, but not HUVECs. Furthermore, it provides the first evidence that farrerol could induce cancer cell growth inhibition by G0/G1-phase cell-cycle arrest mediated by sustained ERK activation. The findings show the potential of farrerol as a chemotherapeutic agent without vascular toxicity for use against gastric cancer.

Synthesis and Biological Activities of New Halophenols

A series of new halophenols were synthesized, and their structures were established on the basis of 1H, 13C NMR and mass spectral data. All of the prepared compounds were screened for their in vitro protein tyrosine kinase (PTK) and vascular smooth muscle cell (VSMC) proliferation inhibitory activity. Twelve halophenols showed significant PTK inhibitory activity, most of them exhibited stronger activities than that of genistein, a positive reference compound. Several halophenols also displayed moderate VSMC proliferation inhibitory activity, compound 8c showed higher activity than that of tetrandrine, a positive reference compound. The preliminary structure-activity relationships of these compounds were investigated and discussed. The results provided a foundation for the action mechanism study and further structure optimization of the halophenols.

Farrerol can attenuate the aortic lesion in spontaneously hypertensive rats via the upregulation of eNOS and reduction of NAD(P)H oxidase activity.

Farrerol, a typical natural flavanone, is the major active component of Rhododendron dauricum L. The objective of this study was to evaluate the attenuation effect of farrerol against the aortic lesions in spontaneously hypertensive rats (SHR) for the first time. Twelve-week-old male SHR were orally administered with farrerol (50mg/kg/day), verapamil (50mg/kg/day, positive control), or vehicle for 8 weeks (n=10 in each group). Age-matched Wistar-Kyoto rats (WKY) served as normal controls (n=10). Our results revealed that farrerol significantly reduced the systolic blood pressure in SHR (from 177±4mmHg to 158±5mmHg) and also dramatically attenuated the aortic lesion, which is characterized by decreased media thickness, wall area, media-lumen ratio, nuclei size and an increased nuclei number (P<0.05). Moreover, the levels of O2(-) along with NAD(P)H oxidase activity were reduced (P<0.05), while the activity of endothelial nitric oxide synthase (eNOS) was elevated (P<0.05) in aortic homogenates after the intervention of farrerol. Furthermore, farrerol upregulated the expression of eNOS in both of mRNA and protein levels, accompanied by the downregulated mRNA and protein expression of p22(phox) (P<0.05), an essential subunit for NADPH oxidase activity. Our findings indicated that farrerol has a significant protective effect against the aortic lesion in SHR, which may be related to the enhanced eNOS activity and reduced NAD(P)H oxidase activity.