The role of the Rho/ROCK signaling pathway in inhibiting axonal regeneration in the central nervous system

The Rho/Rho-associated coiled-coil containing protein kinase（Rho/ROCK） pathway is a major signaling pathway in the central nervous system, transducing inhibitory signals to block regeneration. After central nervous system damage, the main cause of impaired regeneration is the presence of factors that strongly inhibit regeneration in the surrounding microenvironment. These factors signal through the Rho/ROCK signaling pathway to inhibit regeneration. Therefore, a thorough understanding of the Rho/ROCK signaling pathway is crucial for advancing studies on regeneration and repair of the injured central nervous system.

Involvement of Endoplasmic Reticulum Stress in Apoptosis of Testicular Cells Induced by Low-dose Radiation

Summary： The study examined the role of endoplasmic reticulum stress （ERS） and signaling pathways of inositol-requiring enzyme-1 （IRE1）, RNA-activated protein kinase-like ER kinase （PERK） and activating transcription factor-6 （ATF6） in apoptosis of mouse testicular cells treated with low-dose radiation （LDR）. In the dose-dependent experiment, the mice were treated with whole-body X-ray irradiation at different doses （25, 50, 75, 100 or 200 mGy） and sacrificed 12 h later. In the time-dependent experiment, the mice were exposed to 75 mGy X-ray irradiation and killed at different time points （3, 6, 12, 18 or 24 h）. Testicular cells were harvested for experiments. H202 and NO concentrations, and Ca2＋-ATPase activity were detected by biochemical assays, the calcium ion concentration （[Ca2＋]i） by flow cytometry using fluo-3 probe, and GRP78 mRNA and protein expressions by quantitative real-time RT-PCR （qRT-PCR） and Western blotting, respectively. The mRNA expressions of S-XBP1, JNK, caspase-12 and CHOP were measured by qRT-PCR, and the protein expressions of IREla, S-XBP1, p-PERK, p-elF2a, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP by Western blot- ting. The results showed that the concentrations of H202 and NO, the mR_NA expressions of GRP78, S-XBP1, JNK, caspase-12 and CHOP, and the protein expressions of GRP78, S-XBP1, IREla, p-PERK, p-elF2a, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP were significantly increased in a time- and dose-dependent manner after LDR. But the [Ca2]i and Ca2-ATPase activities were sig nificantly decreased in a time and dose-dependent manner. It was concluded that the ERS, regulated by IRE 1, PERK and ATF6 pathways, is involved in the apoptosis of testicular cells in LDR mice, which is associated with ERS-apoptotic signaling molecules of JNK, caspase-12 and CHOP.

Expression and function of Delta-like ligand 4 in a rat model of retinopathy of prematurity

The Delta-like ligand 4/Notch signaling pathway was shown to participate in the process of retinal development and angiogenesis. However, the function of the Delta-like ligand 4/Notch signaling pathway in retinopathy of prematurity requires further study. Retinopathy of prematurity was induced in 5-day-old Sprague-Dawley rats exposed to hyperoxia for 7 days, and then returned to room air. Reverse transcription-PCR and western blot revealed that Delta-like ligand 4 levels decreased at postnatal day 12 and increased at postnatal day 17 in retinopathy of prematurity rats. Flat-mounted adenosine diphosphatase stained retina and hematoxylin-eosin stained retinal tissue slices showed that the clock hour scores and the nuclei counts in retinopathy of prematurity rats were significantly different compared to normal control rats. After retinopathy of prematurity rats were intravitreally injected with Delta-like ligand 4 monoclonal antibody to inhibit the Delta-like ligand 4/Notch signaling pathway, there was a significant increase in the severity of retinal neovascularization （clock hours） in the intravitreally injected eyes. The nuclei count was highly correlated with the clock hour score. These results suggest that Delta-like ligand 4/Notch signaling plays an essential role in the process of physiological and pathological angiogenesis in the retina.

Group Ⅱ p21-activated kinases as therapeutic targets in gastrointestinal cancer

P21-activated kinases(PAKs) are central players in various oncogenic signaling pathways. The six PAK family members are classified into group Ⅰ(PAK1-3) and group Ⅱ(PAK4-6). Focus is currently shifting from group Ⅰ PAKs to group Ⅱ PAKs. Group Ⅱ PAKs play important roles in many fundamental cellular processes, some of which have particular significance in the development and progression of cancer. Because of their important functions, group Ⅱ PAKs have become popular potential drug target candidates. However, few group Ⅱ PAKs inhibitors have been reported, and most do not exhibit satisfactory kinase selectivity and "drug-like" properties. Isoform- and kinase-selective PAK inhibitors remain to be developed. This review describes the biological activities of group Ⅱ PAKs, the importance of group Ⅱ PAKs in the development and progression of gastrointestinal cancer, and smallmolecule inhibitors of group Ⅱ PAKs for the treatment of cancer.

ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expression in vitro

Spontaneous axonal regeneration of neurons does not occur after spinal cord injury because of inhibition by myelin and other inhibitory factors. Studies have demonstrated that blocking the Rho/Rho-kinase （ROCK） pathway can promote neurite outgrowth in spinal cord injury models. In the present study, we investigated neurite outgrowth and neuronal differentiation in neural stem cells from the mouse subventricular zone after inhibition of ROCK in vitro. Inhibition of ROCK with Y-27632 increased neurite length, enhanced neuronal differentiation, and upregulated the expression of two major signaling pathway effectors, phospho-Akt and phospho-mitogen-activated protein kinase, and the Hippo pathway effector YAP. These results suggest that inhibition of ROCK mediates neurite outgrowth in neural stem cells by activating the Hippo signaling pathway.

Amygdalin Ameliorates Liver Fibrosis through Inhibiting Activation of TGF-β/Smad Signaling

Objective:To observe the effect of amygdalin on liver fibrosis in a liver fibrosis mouse model,and the underlying mechanisms were partly dissected in vivo and in vitro.Methods:Thirty-two male mice were randomly divided into 4 groups,including control,model,low-and high-dose amygdalin-treated groups,8 mice in each group.Except the control group,mice in the other groups were injected intraperitoneally with 10%carbon tetrachloride(CCl4)-olive oil solution 3 times a week for 6 weeks to induce liver fibrosis.At the first 3 weeks,amygdalin(1.35 and 2.7 mg/kg body weight)were administered by gavage once a day.Mice in the control group received equal quantities of subcutaneous olive oil and intragastric water from the fourth week.At the end of 6 weeks,liver tissue samples were harvested to detect the content of hydroxyproline(Hyp).Hematoxylin and eosin and Sirius red staining were used to observe the inflammation and fibrosis of liver tissue.The expressions of collagenⅠ(Col-Ⅰ),alpha-smooth muscle actin(α-SMA),CD31 and transforming growth factorβ(TGF-β)/Smad signaling pathway were detected by immunohistochemistry,quantitative real-time polymerase chain reaction and Western blot,respectively.The activation models of hepatic stellate cells,JS-1and LX-2 cells induced by TGF-β1 were used in vitro with or without different concentrations of amygdalin(0.1,1,10μmol/L).The effect of different concentrations of amygdalin on the expressions of liver sinusoidal endothelial cells(LSECs)dedifferentiation markers CD31 and CD44 were observed.Results:High-dose of amygdalin significantly reduced the Hyp content and percentage of collagen positive area,and decreased the mRNA and protein expressions of Col-Ι,α-SMA,CD31 and p-Smad2/3 in liver tissues of mice compared to the model group(P<0.01).Amygdalin down-regulated the expressions of Col-Ⅰandα-SMA in JS-1 and LX-2 cells,and TGFβR1,TGFβR2 and p-Smad2/3 in LX-2 cells compared to the model group(P<0.05 or P<0.01).Moreover,1 and 10μmol/L amygdalin inhibited the mRNA and protein expressions of CD31 in LSECs and increased CD44 expression compared to the model group(P<0.05 or P<0.01).Conclusions:Amygdalin can dramatically alleviate liver fibrosis induced by CCl4 in mice and inhibit TGF-β/Smad signaling pathway,consequently suppressing HSCs activation and LSECs dedifferentiation to improve angiogenesis.

Synergism between Carnosic Acid and Arsenic Trioxide on Induction of Acute Myeloid Leukemia Cell Apoptosis Is Associated with Modulation of PTEN/Akt Signaling Pathway

Objective： To investigate the synergistic effects of carnosic acid （CA） with arsenic trioxide （As203） on proliferation and apoptosis in HL-60 human myeloid leukemia cells, and the major cellular signaling pathway involved in these effects. Methods： HL-60 cellular proliferation was measured by 3-（4,5-dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide （MTF） analysis. Cell cycle distribution and apoptosis were monitored by flow cytometry. The activation of casepase-9, Bcl-2-associated agonist of cell death （BAD）, p-BAD, p27, phosphatase and tensin homolog deleted on chromosome ten （PTEN）, Akt, p-Akt was assessed by Western blot analysis. The expression of PTEN mRNA was tested by reverse transcription polymerase chain reaction （RT-PCR） analysis. Results： CA reduced HL-60 cell viability in a dose- and time-dependent manner, and induced G1 arrest and apoptosis. Moreover, CA upregulated PTEN expression, blocked the Akt signaling pathway, subsequently inhibited phosphorylation of BAD, reactivated caspase-9, and elevated levels of p27. CA also augmented these effects of As203. Conclusion： CA might be a novel candidate of the combination therapy for leukemia treatment; these effects were apparently associated with the modulation of PTEN/Akt signaling pathway.

Biological Function and Mechanism of Long Noncoding RNAs Nuclear-Enriched Abundant Transcript I in Development of Cervical Cancer

Background： Accumulating documents have demonstrated that long noncoding RNAs （lncRNAs） play critical roles in tumorigenesis. As an lncRNA, nuclear-enriched abundant transcript 1 （NEAT1） has been identified to be involved in the progression of many types of cancers. However, the biological function of NE.4T1 in cervical cancer is not fully investigated. The aim of this study was to disclose the specific biological function of lncRNA NEATI in cervical cancer progression. Methods： Quantitative real-time polymerase chain reaction （qRT-PCR） was utilized to identify the expression of lncRNA NE,4 T1 in the cervical cancer tissues and cell lines. All cervical cancer samples used in this study were collected from the Affiliated Suzhou Hospital of Nanjing Medical University between September 2012 and September 2017. The correlation between NE,4T1 expression and the overall survival rate of cervical cancer patients was analyzed by Kaplan-Meier analysis. The effects of NEAT1 knockdown or overexpression on cell proliferation were tested by performing MTT assays and colony formation assays. Transwell assays were conducted to detect the migratory ability of cervical cancer cells, in which NEAT1 was silenced or overexpressed. Western blotting was utilized to validate whether NEAT1 promotes cervical cancer progression through activating PI3K-Akt signaling pathway. Results： High expression of NE,4T1 predicted poor prognosis of cervical cancer patients （χ^2= 0.735, P = 0.005）. Knockdown of NE,4T1 decreased the number of colonies in CaSki cell from 136.667 ± 13.503 to 71.667 ± 7.506 （t = -18.76, P = 0.003） and decreased the number of colonies in HeLa cell from 128.667 ± 13.317 to 65.667 ± 7.024 （t = -5.54, P = 0.031）. However, overexpression of NEA T1 increased the number of colonies in SiHa cell from 84.667 ± 12.014 to 150.667 ± 18.037 （t = 7.27, P = 0.018）. Knockdown of NEAT1 decreased the migratory number of CaSki cell from 100.333 ± 9.866 to 58.333 ± 5.859 （t = -8.08, P = 0.015） and reduced the migratory number in HeLa cell from 123.667± 12.097 to 67.667 ± 7.095 （t = -6.03, P = 0.026）. Overexpression of NEAT1 increased the migratory number of SiHa cell from 127.333 ±16.042 to 231.333 ±31.786 （t = 4.92, P = 0.039）. Conclusion： NEAT1 may exert oncogenic function in cervical cancer and serve as a novel therapeutic target for cervical cancer.

Cell therapy in diabetes: current progress and future prospects

Diabetes mellitus, characterized by the impaired metabolism of insulin secretion in β cells, is becoming one of the most prevalent diseases around the world. Recently, cell replacement based on differentiation of various pluripotent stem cells, including embryonic stern cells, induced pluripo- tent stem cells and multipotent stem cells, such as bone mar- row mesenchymal stem cells, adipose-derived stem cells and gnotobiotic porcine skin-derived stem cells, is becoming a promising therapeutic strategy. Cells derived from pancreatic tissues or other tissues that are relevant to β cell differentiation have also been used as cell source. However, in spite of hopeful experimental results, cell therapy in diabetes still confronts certain obstacles, such as purity of cells, functional differentiation of stem cells and possible tumorigenesis, which, in turn, lead to the seeking of new-generation tools, such as xenogenetic materials. In this review, we will sum- marize the current knowledge and future prospects of cell therapy in diabetes mellitus.

Zhizhu Decoction Alleviates Intestinal Barrier Damage via Regulating SIRT1/FoxO1 Signaling Pathway in Slow Transit Constipation Model Mice

Objective:To explore the possible effects and mechanism of Zhizhu Decoction(ZZD)on the pathophysiology of slow transit constipation(STC).Methods A total of 54 C57BL/6 mice was randomly divided into the following 6 groups by a random number table,including control,STC model(model),positive control,and low-,medium-and high-doses ZZD treatment groups(5,10,20 g/kg,namely L,M-,and H-ZZD,respectively),9 mice in each group.Following 2-week treatment,intestinal transport rate(ITR)and fecal water content were determined,and blood and colon tissue samples were collected.Hematoxylin-eosin and periodic acid-Schiff staining were performed to evaluate the morphology of colon tissues and calculate the number of goblet cells.To determine intestinal permeability,serum levels of lipopolysaccharide(LPS),low-density lipoprotein(LDL)and mannose were measured using enzyme-linked immunosorbent assay(ELISA).Western blot analysis was carried out to detect the expression levels of intestinal tight junction proteins zona-occludens-1(ZO-1),claudin-1,occludin and recombinant mucin 2(MUC2).The mRNA expression levels of inflammatory cytokines including tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,IL-4,IL-10 and IL-22 were determined using reverse transcription-quantitative reverse transcription reaction.Colon indexes of oxidative stress were measured by ELISA,and protein expression levels of colon silent information regulator 1/forkhead box O transcription factor 1(SIRT1/FoxO1)antioxidant signaling pathway were detected by Western blot.Results Compared with the model group,ITR and fecal moisture were significantly enhanced in STC mice in the M-ZZD and H-ZZD groups(P<0.01).Additionally,ZZD treatment notably increased the thickness of mucosal and muscular tissue,elevated the number of goblet cells in the colon of STC mice,reduced the secretion levels of LPS,LDL and mannose,and upregulated ZO-1,claudin-1,occludin and MUC2 expressions in the colon in a dose-dependent manner,compared with the model group(P<0.05 or P<0.01).In addition,ZZD significantly attenuated intestinal inflammation and oxidative stress and activated the SIRT1/FoxO1 signaling pathway(P<0.05 or P<0.01).Conclusion ZZD exhibited beneficial effects on the intestinal system of STC mice and alleviated intestinal inflammation and oxidative stress via activating SIRT1/FoxO1 antioxidant signaling pathway in the colon.

A transposon-introduced G-quadruplex motif is selectively retained and constrained to downregulate CYP321A1

Insects utilize xenobiotic compounds to up-and downregulate cytochrome P450 monooxygenases(P450s)involved in detoxification of toxic xenobiotics including phytochemicals and pesticides.G-quadruplexes(G4)-forming DNA motifs are enriched in the promoter regions of transcription factors and function as cis-acting elements to regulate these genes.Whether and how P450s gain and keep G4 DNA motifs to regulate their expression still remain unexplored.Here,we show that CYP321A1,a xenobiotic-metabolizing P450 from Helicoverpa zea,a polyphagous insect of economic importance,has acquired and preserved a G4 DNA motif by selectively retaining a transposon known as HzIS1-3 that carries this G4 DNA motif in its promoter region.The HzIS1-3 G4 DNA motif acts as a silencer to suppress the constitutive and induced expression of CYP321A1 by plant allelochemicals flavone and xanthotoxin through folding into an intramolecular parallel or hybrid-1 conformation in the absence or presence of K^(+).The G4 ligand N-methylmesoporphyrin IX(NMM)strengthens the silencing effect of HzIS1-3 G4 DNA motif by switching its structure from hybrid-1 to hybrid-2.The enrichment of transposons in P450s and other environment-adaptation genes implies that selective retention of G4 DNA motif-carrying transposons may be the main evolutionary route for these genes to obtain G4 DNA motifs.