Identification of apoptosis-related microRNAs and their target genes in myocardial infarction post-transplantation with skeletal myoblasts.

BACKGROUND: Skeletal myoblasts (SkMs) has provided a promising treatment for myocardial infarction (MI). Functioning as posttranscriptional regulators, microRNAs (miRNAs) play important roles in cardiac repairment and stem cell regulation. However, the correlation between miRNAs and their targeted genes in SkM cell therapy for MI was not fully understood. METHODS: We explored the cardioprotection by SkMs in infracted rats and determined cardiac functions at 4 weeks. In addition, we compared the expression profiles of miRNAs and mRNAs in post-MI rats with or without SkM cell therapy using microarray. The concordance between miRNA expression and mRNA levels of potential target genes was confirmed by quantitative real-time PCR. RESULTS: Quantitative echocardiography and histology showed improved cardiac function, attenuated heart infarcted area and inhibited cardiomyocyte apoptosis in the SkM group, compared with MI group. We identified that 160 miRNAs were differentially expressed in MI group as compared to the control group and 78 miRNAs were differentially expressed in the SkM treated group as compared to the untreated post-MI. We focused on a novel set of apoptosis-associated miRNAs and their target genes, among which 4 miRNAs (miR-30a-5p, miR-30c-5p, miR-145-5p, miR-140-3p), except one (miR-143-3p), were downregulated in the SkM treated group as compared to the untreated group. Furthermore, we found seven genes including Angptl4, Dpep1, Egr1, Eif5a, Tsc22d3, Irs2 and Cebpb that showed a linear correlation with which miRNAs. CONCLUSIONS: The downregulation of apoptosis-regulatory miRNAs and in turn upregulation of target genes may partially account for rescue effect of SKM therapy for MI.

Protective effect of liraglutide on the myocardium of type 2 diabetic rats by inhibiting polyadenosine diphosphate-ribose polymerase-1.

BACKGROUND: In recent years, studies have found that the occurrence and development of diabetic cardiomyopathy (DCM) is closely related to an increase in polyadenosine diphosphate-ribose polymerase-1 (PARP-1) activity. PARP-1 activation could be involved in the pathophysiological process of DCM by promoting oxidative stress, the inflammatory response, apoptosis and myocardial fibrosis. AIM: To investigate the mechanism of liraglutide in improving myocardial injury in type 2 diabetic rats, further clarified the protective effect of liraglutide on the heart, and provided a new option for the treatment of DCM. METHODS: Forty healthy male SD rats aged 6 wk were randomly divided into two groups, a normal control group (n = 10) and a model group (n = 30), which were fed an ordinary diet and a high-sugar and high-fat diet, respectively. After successful modeling, the rats in the model group were fed a high-glucose and high-fat diet for 4 wk and randomly divided into a model group and an intervention group (further divided into a high-dose group and a low-dose group). The rats were fed a high-glucose and high-fat diet for 8 wk and then started drug intervention. Blood samples were collected from the abdominal aorta to detect fasting blood glucose and lipid profiles. Intact heart tissue was dissected, and its weight was used to calculate the heart weight index. Haematoxylin and eosin staining was used to observe the pathological changes in the myocardium and the expression of PARP-1 in the heart by immunohistochemistry. RESULTS: The body weight and heart weight index of rats in the model group were significantly increased compared with those in the normal control group, and those in the intervention group were decreased compared with those in the model group, with a more obvious decrease observed in the high-dose group (P < 0.05). In the model group, myocardial fibers were disordered, and inflammatory cells and interstitial fibrosis were observed. The cardiomyopathy of rats in the intervention group was improved to different degrees, the myocardial fibers were arranged neatly, and the myocardial cells were clearly striated; the improvement was more obvious in the high-dose group. Compared with the normal control group, the expression of PARP-1 in myocardial tissue of the model group was increased, and the difference was statistically significant (P < 0.05). After liraglutide intervention, compared with the model group, the expression of PARP-1 in myocardial tissue was decreased, and the reduction was more obvious in the high-dose group (P < 0.05) but still higher than that in the normal control group. CONCLUSION: Liraglutide may improve myocardial injury in type 2 diabetic rats by inhibiting the expression of myocardial PARP-1 in a dose-dependent manner.

Network pharmacological analysis of corosolic acid reveals P4HA2 inhibits hepatocellular carcinoma progression.

BACKGROUND: Corosolic acid is a pentacyclic triterpene acid with hypoglycemic, anti-inflammatory, and anti-cancer effects. However, its potential targets in hepatocellular carcinoma (HCC) are unknown, hindering clinical utilization. METHODS: Differentially expressed proteins of the Bel-7404 cell line were identified with tandem mass tag analysis and differentially expressed genes (DEGs) of an HCC TCGA dataset using bioinformatics. Gene functions and pathways were inferred using the DAVID database. Online databases were used to establish P4HA2 expression in HCC (GEPIA2) and its relationship with patient survival (UALCAN and The Human Protein Atlas), the association between P4HA2 expression and immune cell infiltration (TIMER2), and DNA methylation of the P4HA2 gene (MethSurv). Cell proliferation, cell cycle, and cell death were assessed with PI and SYTOX-Green staining, CCK-8, and colony formation assays. Protein expression levels were detected by Western blotting. RESULTS: A total of 44 differentially expressed proteins and 4498 DEGs were identified. Four genes whose proteins were also found in the differential protein profile but with opposing expressions were selected as candidate targets. The candidate gene prolyl 4-hydroxylase subunit alpha 2 (P4HA2) was recognized as the only potential target due to its high expression in public datasets, association with poor patient survival, and relation to immune cell infiltration in HCC tissues. Moreover, the DNA methylation status in 4 CpG islands of the P4HA2 gene correlated with a poor prognosis. Furthermore, corosolic acid treatment inhibited the proliferation of HCC cell lines Bel-7404 and HepG2 in a dose-dependent manner, caused G2/M phase cell cycle arrest, and promoted cell death. In addition, the treatment reduced P4HA2 protein levels. CONCLUSION: Our results indicate that P4HA2 is a potential target of corosolic acid. Thus, they contribute to understanding molecular changes in HCC after corosolic acid treatment and facilitate finding new treatment regimens.

MiR-499 induces cardiac differentiation of rat mesenchymal stem cells through wnt/ß-catenin signaling pathway.

OBJECTIVE: To test the hypothesis that over-expressing miR-499 in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) induces them to differentiate into cardiomyocyte-like cells through the wnt/ß-catenin signaling pathway. METHODS: Rat BM-MSCs were infected with lentiviral vectors bearing miR-499. The expression of cardiac-specific markers, NKx2.5, GATA4, MEF2C, and cTnI in these cells were examined by rtPCR or Western blot analysis and the activity of the wnt/ß-catenin signaling pathway was evaluated by measuring the phosphorylation status of ß-catenin. RESULTS: Over-expression of miR-499 in rat BM-MSCs increased the expression of cardiac-specific genes, such as NKx2.5, GATA4, MEF2C, and cTnI and decreased the ratio of phosphorylated/dephosphorylated ß-catenin in the wnt/ß-catenin signaling pathway, thus activating the pathway. Knocking down the expression of Dvl, an adaptor molecule in the wnt/ß-catenin signaling, partially blocked the role of the miR-499 and decreased those cardiac-specific genes. CONCLUSION: Over-expression of miR-499 in rat BM-MSCs induces them toward cardiac differentiation through the activating the wnt/ß-catenin signal pathway.

Effect of liraglutide on endoplasmic reticulum stress in the renal tissue of type 2 diabetic rats.

BACKGROUND: Liraglutide is a glucagon-like peptide 1 receptor agonist analog that has been found to have a therapeutic effect in diabetes. In addition to its ability to treat diabetes, liraglutide has beneficial effects on the cardiovascular system and kidney as well as other beneficial effects, but its specific mechanism is not clear. In this study, a rat model of type 2 diabetes was established by administration of a high-sugar, high-fat diet combined with low-dose streptozotocin (STZ) to observe the effect of liraglutide on the kidneys of type 2 diabetes rats and the possible underlying mechanisms. AIM: To explore whether liraglutide has a protective effect on type 2 diabetic rat kidneys and the underlying mechanisms. METHODS: Eight-week-old male Sprague-Dawley rats were randomly divided into a control group, model group, low-dose liraglutide group, and high-dose liraglutide group. Control rats were fed a standard diet, while model group and intervention group rats were fed high-sugar, high-fat feed for 1 mo and then intraperitoneally injected with 40 mg/kg STZ to induce type 2 diabetes. The low-dose and high-dose intervention groups received 100 µg/kg and 200 µg/kg liraglutide, respectively, once daily by subcutaneous injection. The control and model groups were given an equivalent volume of physiological saline for 8 wk. Pathological changes in renal tissues were observed by hematoxylin and eosin staining and periodic acid-Schiff staining, and GRP78 and caspase-12 expression was detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Western blot analysis showed that GRP78 and caspase-12 protein expression in kidney tissue was significantly higher in model rats than in normal rats and lower in the liraglutide-treated groups than in the model group, with a more significant decrease being observed in the high-dose group than in the low-dose group. RT-PCR showed that the mRNA expression of GRP78 and caspase-12 was higher in model rats than in control rats and lower in the liraglutide-treated groups than in the model group, with the high-dose group exhibiting a more significant decrease than the low-dose group. CONCLUSION: Liraglutide may delay the progression of diabetic nephropathy by reducing endoplasmic reticulum stress and protect the kidneys in a dose-dependent manner.

Impacts of sterilization, microwave and ultrasonication pretreatment on hydrogen producing using waste sludge.

Hydrogen production by sterilization, microwave and ultrasonication pretreated waste sludge was investigated in this study. A new strain of Pseudomonas sp. GZ1 (EF551040) was inoculated in pretreated waste sludge to produce hydrogen. The experimental results showed that different pretreated sludge had evident differences in the yield of hydrogen production and lag time. Sterilized sludge had the largest yield of hydrogen production, and the maximum yield was 15.02 ml/gTCOD. The lag time of using sterilized sludge was 15 h, longer than other two pretreated sludge. Using the ultrasonicated sludge, the hydrogen production yield was smallest and lag time was shortest in the three pretreated sludge. Protein and carbohydrate could be released from waste sludge by pretreatment. Protein was the main nutrient used for hydrogen production. The concentration of protein, carbohydrate and SCOD increased after pretreatment and fermentation. The impacts of different pretreatments on hydrogen production were also discussed in detail.

Total concentrations and speciation of heavy metals in municipal sludge from Changsha, Zhuzhou and Xiangtan in middle-south region of China.

The presence of heavy metals in municipal sludge restricts its use for agricultural purposes. In this paper, the bioavailability and eco-toxicity of heavy metals in municipal sludge was evaluated, taking into consideration both the speciation of metals and the local environmental characteristics. The dewatered municipal sludge samples were collected from five sewage plants in Changsha, Zhuzhou and Xiangtan respectively, which are representative cities with characteristics of the middle-south region of China. Some agricultural significant parameters and total metal concentrations in the sludge were determined and the metal speciation was studied by using BCR sequential extraction procedure. It was found experimentally that in general the municipal sludge collected from the five sewage plants was rich in organics, N and P. Except that the sludge from Xia Wan Sewage Treatment Plant showed higher concentrations of heavy metals, the sludge from other plants all showed a low total content of heavy metals with only Cd slightly exceeding the permitted values of the national application standard of acid soil in China (GB18918-2002). The sequential extraction results showed that Cu and Zn were principally distributed in the oxidizable fraction, which meant a high potential toxicity, but the bioavailability of Zn might be overestimated to the soil of Hunan. Pb was mainly in the residual fraction. The distribution of Cd showed no obvious characteristics.

Adsorption of Cd(II) and Cu(II) from aqueous solution by carbonate hydroxylapatite derived from eggshell waste.

Carbonate hydroxylapatite (CHAP) synthesized by using eggshell waste as raw material has been investigated as metal adsorption for Cd(II) and Cu(II) from aqueous solutions. The effect of various parameters on adsorption process such as contact time, solution pH, amount of CHAP and initial concentration of metal ions was studied at room temperature to optimize the conditions for maximum adsorption. The results showed that the removal efficiency of Cd(II) and Cu(II) by CHAP could reach 94 and 93.17%, respectively, when the initial Cd(II) concentration 80 mg/L and Cu(II) 60 mg/L and the liquid/solid ratio was 2.5 g/L. The equilibrium sorption data for single metal systems at room temperature could be described by the Langmuir and Freundlich isotherm models. The highest value of Langmuir maximum uptake, (b), was found for cadmium (111.1mg/g) and copper (142.86 mg/g). Similar Freundlich empirical constants, K, were obtained for cadmium (2.224) and copper (7.925). Ion exchange and surface adsorption might be involved in the adsorption process of cadmium and copper. Desorption experiments showed that CaCl2, NaCl, acetic acid and ultrasonic were not efficient enough to desorb substantial amount of metal ions from the CHAP. The results obtained show that CHAP has a high affinity to cadmium and copper.

A two-stage anaerobic system for biodegrading wastewater containing terephthalic acid and high strength easily degradable pollutants.

The high strength easily biodegradable pollutants (represented by CODE) are strong inhibitors of terephthalic acid (TA) anaerobic biodegradation. At the same time, TA can inhibit easily biodegradable pollutants removal under anaerobic conditions to a limited extent. This mutual inhibition could happen and cause a low removal efficiency of both TA and CODE, when the effluent from TA workshops containing TA and easily biodegradable pollutants are treated by a single anaerobic reactor system. Based upon the treatment kinetics analysis of both TA degradation and CODE removal, a two-stage up-flow anaerobic sludge blanket and up-flow fixed film reactor(UASB-UAFF) system for dealing with this kind of wastewater was developed and run successfully at laboratory scale. An UASB reactor with the methanogenic consortium as the first stage removes the easily biodegradable pollutants(CODE). An UAFF reactor as the second stage is mainly in charge of TA degradation. At a CODE loading of 15.3 g/(L.d) and a TA loading of 1.4 g/(L.d), HRT 18.5 h, the CODE and TA removal rate of the system reached 89.2% and 71.6%, respectively.

Novel mechanism of inhibition of dendritic cells maturation by mesenchymal stem cells via interleukin-10 and the JAK1/STAT3 signaling pathway.

Mesenchymal stem cells (MSCs) can suppress dendritic cells (DCs) maturation and function, mediated by soluble factors, such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E(2) (PGE(2)), and nitric oxide (NO). Interleukin-10 (IL-10) is a common immunosuppressive cytokine, and the downstream signaling of the JAK-STAT pathway has been shown to be involved with DCs differentiation and maturation in the context of cancer. Whether IL-10 and/or the JAK-STAT pathway play a role in the inhibitory effect of MSCs on DCs maturation remains controversial. In our study, we cultured MSCs and DCs derived from rat bone marrow under different culturing conditions. Using Transwell plates, we detected by ELISA that the level of IL-10 significantly increased in the supernatants of MSC-DC co-cultures at 48 hours. The cell immunofluorescence assay suggested that the MSCs secreted more IL-10 than the DCs in the co-cultures. Adding exogenous IL-10 to the DCs monoculture or MSC-DC co-cultures stimulated IL-10 and led to a decrease in IL-12, and lower expression of the DCs surface markers CD80, CD86, OX62, MHC-II and CD11b/c. Supplementing the culture with an IL-10 neutralizing antibody (IL-10NA) showed precisely the opposite effect of adding IL-10. Moreover, we demonstrated that the JAK-STAT signaling pathway is involved in inhibiting DCs maturation. Both JAK1 and STAT3 expression and IL-10 secretion decreased markedly after adding a JAK inhibitor (AG490) to the co-culture plate. We propose that there is an IL-10 positive feedback loop, which may explain our observations of elevated IL-10 and enhanced JAK1 and STAT3 expression. Overall, we demonstrated that MSCs inhibit the maturation of DCs through the stimulation of IL-10 secretion, and by activating the JAK1 and STAT3 signaling pathway.

[Enhanced hydrolysis of excess sludge by external enzymes].

The effects of external enzymes to enhance excess sludge hydrolysis were investigated. Protease and alpha-amylase were added singly and in combination, and it was found that external enzymes can enhance sludge hydrolysis. When treated with single enzyme, alpha-amylase had higher efficiency than protease, SCOD/TCOD rose from 16.3% to 22.3% and VSS reduction improved from 39.8% to 54.24% at the optimal dosage of 60 mg/g (calculated by addition of enzyme in TS). Furthermore, mixture enzymes showed better hydrolysis efficiency than single enzyme. The highest VSS reduction was observed at an enzyme mixture ratio of 1:3 with protease and amylase when the hydrolysis temperature was 50 degrees C, and the VSS reduction achieved 68.43%. Correspondingly, the concentration of reducing sugar and NH4+ -N rose from 37.29, 47.60 mg/L to 177.8, 143.43 mg/L, respectively. According to the kinetic and enzymatic analysis of hydrolysis process, sludge hydrolysis within prior 4 h was consistent with first-order reaction dynamic. During the hydrolysis, both protease and amylase activity rose gradually, the highest activity observed were 2.57 U/mL and 4.64 U/mL at 4h after hydrolysis. Thereafter, the enzyme activity decreased.

Enhanced efficiency of biological excess sludge hydrolysis under anaerobic digestion by additional enzymes.

In this investigation, the effects of commercial enzyme preparation containing alpha amylase and neutral protease on hydrolysis of excess sludge and the kinetic analysis of hydrolysis process were evaluated. The results indicated that amylase treatment displayed higher hydrolysis efficiency than that of protease. VSS reduction greatly increased to 39.70% for protease and 54.24% for amylase at the enzyme dosage of 6% (w/w), respectively. The hydrolysis rate of sludge improved with temperature increasing from 40 to 50 degrees Celsius, which could be well described by the amended Arrhenius equation. Mixed-enzyme had great impact on sludge solubilisation than single enzyme. The mixture of two enzymes (protease:amylase=1:3) resulted in optimum hydrolysis efficiency, the efficiency of solids hydrolysis increased from 10% (control test) to 68.43% at the temperature of 50 degrees Celsius. Correspondingly, the concentration of reducing sugar and NH(4)(+)-N improved about 377% and 201%, respectively. According to the kinetic analysis of enzymatic hydrolysis process, VSS solubilisation process within prior 4 h followed first-order kinetics. Compared with control test, the hydrolysis rate improved significantly at 50 degrees Celsius when either single enzyme or mixed-enzyme was added.

[Enrichment and dynamic analysis of deammonification granules].

Deammonification process was started-up in a sequencing batch reactor (SBR) seeded with anaerobic granular sludge. A kinetic model describing deammonification process was proposed. Due to the presence of concentration gradient of dissolved oxygen (DO) in granular sludge, model correction factors of DO were introduced. The influences of denitrification process, nitrite and DO on granular sludge activity were studied by model and good agreement was obtained between the experimental results and simulation results. Heterotrophic denitrifiers has some influence on the ANAMMOX process during the initial cultivation, and the influence decreases gradually during the following operation period. When nitrite concentration is 20-30 mg/L, a total nitrogen removal efficiency begins to drop caused by inhibition of nitrite. With high or low DO concentration, the activity of deammonification is inhibited. A total nitrogen removal efficiency can simultaneously reach higher levels by controlling appropriate DO related NH4(+) -N concentration of inflow. In a NH4(+) -N concentration of 80 mg/L, the optimal DO level for a maximal nitrogen gas production is at 0.3-0.6 mg/L.

[Relationship between interleukin-6 gene-572C/G polymorphism and chronic periodontitis].

OBJECTIVE: To investigate the relationship between interleukin-6 (IL-6) gene -572C/G polymorphism and the risk of chronic periodontitis disease. METHODS: IL-6-572C/G genotype polymorphism was examined in 93 patients with chronic periodontitis and 96 control subjects by PCR restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: IL-6-572GG genotype and G allele were more frequent in chronic periodontitis patients than that in control subjects (P<0.05). -572CC, CG and GG genotype frequency were 52.7 % , 40.9 %, 6.4 % in chronic periodontitis patients and 67.7 %, 31.3%, 1.0 % in control subjects, respectively. -572CG + GG genotype and G allele were more frequent in patients than that in control subjects (P<0.05). Compared with CC genotype,the odds ratio for chronic periodontitis was 1.88 (95% CI: 1.04-3.40, P<0.05) for CG + GG genotype. CONCLUSIONS: The frequency of -572CG + GG genotype was higher in patients with chronic periodontitis compared with the control subjects. IL-6-572C/G polymorphism may be a genetic susceptibility factor for chronic periodontitis in Chinese Hans population.

[Bio-hydrogen production from different pretreated sludge by Pseudomonas sp. GL1 and changes in the liquid phases].

Batch tests of anaerobic fermentative hydrogen production by Pseudomonas sp. GL1 were investigated using sterilization, microwave and ultrasonication pretreated sludge as substrate. The profiles of soluble COD, protein, carbohydrate and pH value during the fermentation process were monitored. The results showed that only hydrogen and carbon dioxide were produced and methane was not observed during the process. A maximal hydrogen yield (30.07 mL x g(-1)) and bio-hydrogen content (81.45%) were obtained from the sterilization pretreated sludge run. The shortest lag time for hydrogen production was in ultrasonication pretreated sludge run (3 h), while the longest one was in sterilization pretreated sludge run (15 h), and the medial one was in microwave pretreated sludge run (12 h). It was found that the changes of sludge substrates (soluble COD, protein, carbohydrate and pH value) were various with different pretreated sludge during the fermentation process, especially in the sterilization sludge run, which implied that the pretreatment method could affect substrate utilization by Pseudomonas sp. GL1.

[Simultaneous nitrogen and phosphorus removal in the reactor of inner loop SBR without anaerobic phase].

The performance of simultaneous nitrogen and phosphorus removal with an inner loop sequencing batch reactor were investigated by simulated municipal wastewater. The experimental results showed that COD, NH4(+) -N, and TP can be removed efficiently after four hours aeration, during which dissolved oxygen concentration was at 6 mg/L at the beginning of aerobic phase and pH was in the rang of 7 - 8. The COD and NH4+ -N as well as TP concentration in the effluent were about at 4 - 48 mg x L(1), 0 - 2.0 mg x L(-1), and 0 - 1.4 mg x L(-1) respectively, which indicated the removal rate for each item were about 89.7% +/- 6.5%, 97.4% +/- 3.6%, 95.6% +/- 4.4% when the concentration of influent were about 170 - 260 mg x L(-1), 20 - 30 mg- L(-1), 8 - 20 mg L(-1), respectively. The removal rate of TIN( NH4 -N + NO3(-) -N + NO2(-) -N) was also reached about 70%. It was found during the research process that phosphorus removal can be achieved without anaerobic phase, which was conventionally considered as a key phase for phosphorus removal, and this phenomena can not be explained by traditional theory.