Perspective of future drugs targeting sterile 20/SPS1-related proline/alanine-rich kinase for blood pressure control

According to a genome-wide association study,intronic SNPs within the human sterile 20/SPS1-related proline/alanine-rich kinase(SPAK) gene was linked to 20% of the general population and may be associated with elevated blood pressure. As cell volume changes,mammalian SPAK kinases respond to phosphorylate and regulate cation-coupled chloride co-transporter activity. To our knowledge,phosphorylation of upstream with-no-lysine(K)(WNK) kinases would activate SPAK kinases. The activation of WNK-OSR1/SPAK cascade on the kidneys and aortic tissue is related to the development of hypertension. Several regulators of the WNK pathway such as the Kelch kinase protein 3-Cullin 3 E3 ligase,hyperinsulinemia,and low potassium intake to mediate hypertension have been identified. In addition,the SPAK kinases may affect the action of renin-angiotensin-aldosterone system on blood pressure as well. In 2010,two SPAK knock-in and knock-out mouse models have clarified the pathogenesis of lowering blood pressure by influencing the receptors on the kidneys and aortic smooth muscle. More recently,two novel SPAK inhibitors for mice,Stock 1S-14279 and Closantel were discovered in 2014. Targeting of SPAK seems to be promising for future antihypertensive therapy. Therefore we raised some viewpoints for the issue for the antihypertensive therapy on the SPAK(gene or kinase).

Effects of tyrosine kinase inhibitor E7080 and eNOS inhibitor L-NIO on colorectal cancer alone and in combination

Objective：To investigate the effects of E7080 and N5-（1-iminoethyl）-L-ornithine dihydrochloride （L-NIO）on colorectal cancer alone and in combination.Methods：HT29 colorectal cancer cell line from Sap Institute was used.Real-time cell analysis （xCELLigence system） was performed to determine the effects of E7080 and L-NIO on colorectal cell proliferation.While apoptosis was determined with Annexin V staining,and the effect of agents on angiogenesis was determined with chorioallantoic membrane （CAM） model.Results：We found that E7080 has a strong antiproliferative effect with an half maximum inhibition of concentration （IC50） value of 5.60×10-8 mol/L.Also it has been observed that E7080 showed antiangiogenic and apoptotic effects on HT29 colorectal cancer cells.Antiangiogenic scores of E7080 were 1.2,t.0 and 0.6 for 100,10 and 1 nmol/L E7080 concentrations,respectively.Furthermore,apoptosis has been detected in 71％ of HT29 colorectal cancer cells after administration of 100 nmol/L E7080 which may indicate strong apoptotic effect.Meanwhile administration of L-NIO alone did not show any effect,but the combination of E7080 with L-NIO increased the antiproliferative,antiangiogenic and apoptotic effects of E7080.Conclusions：Results of this study indicate that E7080 may be a good choice in treatment of colorectal tumors.Furthermore the increased effects of E7080 when combined with L-NIO raise the possibility to use a lower dose of E7080 and therefore avoid/minimize the side effects observed with E7080.

血管生成素-1对SW1116细胞的作用及其机制

目的探讨血管生成素-1(Angiopoietin-1,Ang-1)蛋白对无血清DMEM培养基培养的结肠癌细胞(SW 1116)存活率的影响及其与PI3-′k inase/Akt通路的关系。方法将不同浓度的Ang-1蛋白作用于SW 1116细胞,用MTT法检测细胞增殖,根据实验结果选定Ang-1蛋白的后续实验浓度,设计出SW 1116细胞增殖抑制模型,分别向该模型中加入Ang-1及LY294002,应用免疫印记法分析相关蛋白(Tie-2、PI3K、Akt)的变化。结果 Ang-1组与无血清DMEM培养基组比较,Tie-2、PI3K、Akt三种蛋白在SW 1116细胞中的表达均增强,但仅Tie-2的表达有显著差异(P<0.01),LY294002组三种蛋白的表达均减弱(P<0.01,P<0.05)。结论较低浓度(0.05 mg/L)的Ang-1蛋白在结肠癌细胞中即有抗凋亡作用,且随着浓度的增加抗凋亡作用逐渐加强,当高于0.2 mg/L时,作用逐渐减弱,其诱导凋亡的机制可能与Tie-2/PI3-′k inase/Akt调节的通路有关,应用该途径的抑制剂LY294002可抑制结肠癌细胞的生长,实现抗肿瘤作用。

Involvement of ERK1/2 and p38 MAPK in up-regulation of 14-3-3 protein induced by hydrogen peroxide preconditioning in PC12 cells

Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 4′,6′-diamidino-2-phenylindole （DAPI） staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase （MAPK） were determined by Western blot. Enzyme-linked immunosorbent assay （ELISA） was used to measure the activity of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^＋ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^＋- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.

Neuroprotective effects of exogenous brain-derived neurotrophic factor on amyloid-beta 1-40-induced retinal degeneration

Amyloid-beta(Aβ)-related alterations,similar to those found in the brains of patients with Alzheimer's disease,have been observed in the retina of patients with glaucoma.Decreased levels of brain-derived neurotrophic factor(BDNF)are believed to be associated with the neurotoxic effects of Aβpeptide.To investigate the mechanism underlying the neuroprotective effects of BDNF on Aβ_(1-40)-induced retinal injury in Sprague-Dawley rats,we treated rats by intravitreal administration of phosphate-buffered saline(control),Aβ_(1-40)(5 nM),or Aβ_(1-40)(5 nM)combined with BDNF(1μg/mL).We found that intravitreal administration of Aβ_(1-40)induced retinal ganglion cell apoptosis.Fluoro-Gold staining showed a significantly lower number of retinal ganglion cells in the Aβ_(1-40)group than in the control and BDNF groups.In the Aβ_(1-40)group,low number of RGCs was associated with increased caspase-3 expression and reduced TrkB and ERK1/2 expression.BDNF abolished Aβ_(1-40)-induced increase in the expression of caspase-3 at the gene and protein levels in the retina and upregulated TrkB and ERK1/2 expression.These findings suggest that treatment with BDNF prevents RGC apoptosis induced by Aβ_(1-40)by activating the BDNF-TrkB signaling pathway in rats.

Geniposide, the component of the Chinese herbal formula Tongluojiunao, protects amyloid-β peptide(1–42)-mediated death of hippocampal neurons via the non-classical estrogen signaling pathway

Tongluojiunao （TLJN） is an herbal medicine consisting of two main components, geniposide and ginsenoside Rg1. TLJN has been shown to protect primary cultured hippocampal neurons. How-ever, its mechanism of action remains unclear. In the present study, primary cultured hippocampal neurons treated with Aβ1-42 （10 μmol/L） signiifcantly increased the release of lactate dehydroge-nase, which was markedly reduced by TLJN （2 μL/mL）, speciifcally by the component geniposide （26 μmol/L）, but not ginsenoside Rg1 （2.5 μmol/L）. hTe estrogen receptor inhibitor, ICI182780 （1 μmol/L）, did not block TLJN-or geniposide-mediated decrease of lactate dehydrogenase under Aβ1-42-exposed conditions. However, the phosphatidyl inositol 3-kinase or mitogen-activated protein kinase pathway inhibitor, LY294002 （50 μmol/L） or U0126 （10 μmol/L）, respectively blo cked the decrease of lactate dehydrogenase mediated by TLJN or geniposide. hTerefore, these results suggest that the non-classical estrogen pathway （i.e., phosphatidyl inositol 3-kinase or mitogen-activated protein kinase） is involved in the neuroprotective effect of TLJN, speciifcally its component, geniposide, against Aβ1-42-mediated cell death in primary cultured hippocampal neurons.

保罗样激酶PLK1与肿瘤预后及治疗研究进展

保罗样激酶1(PLK1)在启动、维持及完成有丝分裂中扮演重要角色。已发现PLK1基因在多种恶性肿瘤中存在过表达,并与某些肿瘤的恶性生物学行为及预后密切相关,有望成为恶性肿瘤的又一新标记物及肿瘤治疗的新靶点。本文对PLK1与肿瘤预后及治疗相关研究进展做一综述。

MiR-32-5p aggravates intestinal epithelial cell injury in pediatric enteritis induced by Helicobacter pylori

BACKGROUND Pediatric enteritis is one of the infectious diseases in the digestive system that causes a variety of digestive problems,including diarrhea,vomiting,and bellyache in children.Clinically,Helicobacter pylori(H.pylori)infection is one of the common factors to cause pediatric enteritis.It has been demonstrated that aberrant expression of microRNAs(miRNAs)is found in gastrointestinal diseases caused by H.pylori,and we discovered a significant increase of miR-32-5p in H.pylori-related pediatric enteritis.However,the exact role of miR-32-5p in it is still unknown.AIM To investigate the role of aberrant miR-32-5p in pediatric enteritis induced by H.pylori.METHODS MiR-32-5p expression was detected by quantitative real time-polymerase chain reaction.The biological role of miR-32-5p in H.pylori-treated intestinal epithelial cells was evaluated by Cell Counting Kit-8 assay and flow cytometry.The potential target of miR-32-5p was predicted with TargetScanHuman and verified by luciferase assay.The downstream mechanism of miR-32-5p was explored by using molecular biology methods.RESULTS We found that miR-32-5p was overexpressed in serum of H.pylori-induced pediatric enteritis.Further investigation revealed that H.pylori infection promoted the death of intestinal epithelial cells,and increased miR-32-5p expression.Moreover,miR-32-5p mimic further facilitated apoptosis and inflammatory cytokine secretion of intestinal epithelial cells.Further exploration revealed that SMAD family member 6(SMAD6)was the direct target of miR-32-5p,and SMAD6 overexpression partially rescued cell damage induced by H.pylori.The following experiments showed that miR-32-5p/SMAD6 participated in the apoptosis of intestinal epithelial cells induced by transforming growth factor-β-activated kinase 1(TAK1)-p38 activation under H.pylori infection.CONCLUSION Our work uncovered the crucial role of aberrant expression of miR-32-5p in H.pylori–related pediatric enteritis,and suggested that the TAK1-p38 pathway is involved in it.

Effect of DRB on the Biological Characteristics of Human Laryngeal Carcinoma Hep-2 Cell Line

In order to study the effect of 5, 6-Dichloro-l-13-D-ribofuranosyl-benzimidazole （DRB） on the biological characteristics of human laryngeal carcinoma Hep-2 cell line in vitro, Hep-2 cells cultured in vitro were treated with different concentrations of DRB. Changes in cell proliferation, apoptotic rate and invasiveness were detected by MTT assay, flow cytometry （FCM） and matrigel in vitro invasion assay, respectively. It was found that DRB inhibited the proliferation of Hep-2 cells in a dose- and time-dependent manner. After being treated with 0, 10, 20, 40, 80 μmmol/L DRB for 24 h, the apoptotic rate in Hep-2 cells was （0.68±0.19）%, （1.95±0.12）%, （8.51±0.26）%, （11.26±0.17）% and （14.99±0.32）%, respectively. The matrigel in vitro invasion assay revealed that DRB began to inhibit the invasion of Hep-2 cells at the concentration of 5 μmmol/L, and with the increase of DRB concentration, the inhibitory effect was enhanced. It was suggested that DRB could influence the essential biological characteristics of Hep-2 cells, inhibit Hep-2 cells proliferation, reduce invasive ability and induce apoptosis of Hep-2 cells.

PI-3 kinase pathway can mediate the effect of TGF-β1 in inducing the expression of SHARP-2 in LLC-PK1 cells

We aim to investigate the effect of transforming growth factor （TGF）-β1 on the expression of enhancer of split- and hairy-related protein-2 （SHARP-2） messenger RNA （mRNA） and its signaling pathway. In this study, several cell lines including LLC-PK1 （a porcine kidney tubular epithelial cell line）, MDCK （Madin-Darby canine kidney） and CTLL-2 （cytotoxic T-lymphocyte line） were treated with recombinant human TGF-131, and a series of experiments were carried out, involving Northern blot analysis of total RNA from these cells. Further, several specific chemical inhibitors were applied before TGF-β1 treatment to probe the signaling pathway. The results showed that TGF-β1 can significadtly up-regulate SHARP-2 mRNA expression in the LLC-PK1 cell line. The peak level of induction was found 2 h after TGF-β1 stimulation. While one phospho- inositide 3-kinases （PI-3） kinase inhibitor, LY294002, completely blocked the effect of TGF-131 on SHARP-2 mRNA expression in LLC-PK1 cells at a low concentration, other inhibitors, including PD98059, staurosporine, AG490, wortmannin, okadaic acid and rapamycin, had no effect. The effect of LY294002 was dose-dependent. We conclude that, in LLC-PK1 cells at least, TGF-β1 can effectively induce the SHARP-2 mRNA expression and that the PI-3 kinase pathway can mediate this effect.

Regression of cardiovascular remodeling in hypertension:Novel relevant mechanisms

Asymptomatic organ damage due to progressive kidney damage, cardiac hypertrophy and remodeling put hypertensive patients at high risk for developing heart and renal failure, myocardial infarction and stroke. Current antihypertensive treatment normalizes high blood pressure, partially reverses organ damage, and reduces the incidence of heart and renal failure. Activation of the renin-angiotensin system(RAS) is a primary mechanism of progressive organ damage and, specifically, a major cause of both renal and cardiovascular fibrosis. Currently, inhibition of the RAS system [mainly with angiotensin I converting enzyme inhibitors or angiotensin II(Ang II) receptor antagonists] is the most effective antihypertensive strategy for normalizing blood pressure and preventing target organ damage. However, residual organ damage and consequently high risk for cardiovascular events and renal failure still persist. Accordingly, in hypertension, it is relevant to develop new therapeutic perspectives, beyond reducing blood pressure to further prevent/reduce target organ damage by acting on pathways that trigger and maintain cardiovascular and renal remodeling. We review here relevant novel mechanisms of target organ damage in hypertension, their role and evidence in prevention/regression of cardiovascular remodeling and their possible clinical impact as well. Specifically, we focus on the signaling pathway Rho A/Rho kinase, on the impact of the vasodilatory peptides from the RAS and some insights on the role of estrogens and myocardial chymase in cardiovascular hypertensive remodeling.

Study on the relationship between relieving energy crisis in myofascial trigger points with An-Pressing manipulation and AMPK/PGC-1α pathway activation

Objective To explore the mechanism of An-Pressing manipulation in relieving energy crisis in chronic myofascial trigger points(MTrPs)by observing the effects of An-Pressing manipulation on adenosine triphosphate(ATP),adenosine 5′-monophosphate(AMP)-activated protein kinase(AMPK)/peroxisome proliferator-activated receptorγcoactivator 1α(PGC-1α)pathway and mitochondrial ultrastructure of skeletal muscle cells in MTrPs rats.Methods Forty-eight male Sprague-Dawley rats were randomly divided into a blank group,a model group,a lidocaine group,and an An-Pressing manipulation group,with 12 rats in each group.The model group,lidocaine group and An-Pressing manipulation group were used to replicate the MTrPs rat model by blunt shock and centrifugal motion method.After modeling,the An-Pressing manipulation group was subjected to 7 times An-Pressing manipulation,once every other day;the lidocaine group was treated with 3 times of injection of lidocaine at the MTrPs,once every 6 d.The blank group and the model group were fed normally without intervention.After the intervention,local muscle tissue was taken to detect the content of ATP and the expression of AMPK,phosphorylated AMPK(phospho-AMPK),PGC-1α,and glucose transporter 4(GluT4),and the ultrastructure of mitochondria was observed under an electron microscope.Results Compared with the blank group,the ATP content in the model group was decreased(P<0.05),the protein expression levels of phospho-AMPK,PGC-1α,and GluT4 and the ratio of phospho-AMPK to AMPK were decreased(P<0.05);under the electron microscope,the number of mitochondria decreased,and they were deformed,small in volume,and had deformed cristae.Compared with the model group,the ATP contents in the An-Pressing manipulation group and the lidocaine group were increased(P<0.05),and the protein expression levels of phospho-AMPK,PGC-1α,and GluT4 and the ratio of phospho-AMPK to AMPK were increased(P<0.05);under the electron microscope,the number of mitochondria increased,the shape and size of the mitochondria were basically normal,and the cristae could be seen.Compared with the lidocaine group,phospho-AMPK and the ratio of phospho-AMPK to AMPK in the An-Pressing manipulation group were increased(P<0.05);under the electron microscope,the numbers of mitochondria were similar,and the shape and size of the mitochondria were basically normal without swelling,and the cristae could be observed.Conclusion An-Pressing manipulation can increase the ATP content in MTrPs tissue,improve the expression levels of PGC-1α and GluT4 proteins and the ratio of phospho-AMPK to AMPK;its mechanism may relate to the activation of AMPK/PGC-1α signaling pathway to promote the repair of mitochondrial damages.

AAZ2 induces mitochondrial-dependent apoptosis by targeting PDK1 in gastric cancer

Drastic surges in intracellular reactive oxygen species(ROS)induce cell apoptosis,while most chemotherapy drugs lead to the accumulation of ROS.Here,we constructed an organic compound,arsenical N-(4-(1,3,2-dithiarsinan-2-yl)phenyl)acrylamide(AAZ2),which could prompt the ROS to trigger mitochondrial-dependent apoptosis in gastric cancer(GC).Mechanistically,by targeting pyruvate dehydrogenase kinase 1(PDK1),AAZ2 caused metabolism alteration and the imbalance of redox homeostasis,followed by the inhibition of phosphoinositide-3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway and leading to the activation of B-cell lymphoma 2(Bcl2)/Bcl2-associated X(Bax)/caspase-9(Cas9)/Cas3 cascades.Importantly,our in vivo data demonstrated that AAZ2 could inhibit the growth of GC xenograft.Overall,our data suggested that AAZ2 could contribute to metabolic abnormalities,leading to mitochondrial-dependent apoptosis by targeting PDK1 in GC.

Phototropism in land plants： Molecules and mechanism from light perception to response

BACKGROUND： Phototropism is the response a plant exhibits when it is faced with a directional blue light stimulus. Though a seemingly simple differential cell elongation response within a responding tissue that results in organ curvature, phototropism is regulated through a complex set of signal perception and transduction events that move from the plasma membrane to the nucleus. In nature phototropism is one of several plant responses that have evolved to optimize photosynthesis and growth. OBJECTIVE： In the present work we will review the state of the field with respect to the molecules and mechanisms associated with phototropism in land plants. METHODS： A systematic literature search was done to identify relevant advances in the field. Though we tried to focus on literature within the past decade （1998-present）, we have discussed and cited older literature where appropriate because of context or its significant impact to the present field. Several previous review articles are also cited where appropriate and readers should seek those out. RESULTS： A total of 199 articles are cited that fulfill the criteria listed above. CONCLUSIONS： Though important numerous and significant advances have been made in our understanding of the molecular, biochemical, cell biological and physiologic mechanisms underlying phototropism in land plants over the past decade, there are many remaining unanswered questions. The future is indeed bright for researchers in the field and we look forward to the next decade worth of discoveries.