Luteolin inhibits subretinal fibrosis and epithelial-mesenchymal transition in laser-induced mouse model via suppression of Smad2/3 and YAP signaling.

BACKGROUND: Subretinal fibrosis (SF) accounts for vision loss in patients with neovascular age-related macular degeneration (nAMD) even treated with adequate intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) drugs. Currently, there is no treatment available to prevent or treat SF caused by nAMD. PURPOSE: This study aims to investigate the potential effects of luteolin on SF and epithelial-mesenchymal transition (EMT) as well as the underlying molecular pathways both in vivo and in vitro. METHODS: Seven-week-old male C57BL/6J mice were employed to establish laser-induced choroidal neovascularization (CNV) and SF. One day after the laser induction, luteolin was administered intravitreally. SF and CNV were assessed with the immunolabeling of collagen type I (collagen I) and isolectin B4 (IB4), respectively. RPE65 and α-SMA colocalization in the lesions was used to evaluate the extent of EMT in retinal pigment epithelial (RPE) cells by using immunofluorescence. In vitro, luteolin was administered to TGFß1-treated primary human RPE (phRPE) cells. RT-qPCR, Western blot and immunofluorescence were employed to evaluate the change of EMT-related molecules, epithelial markers, and relevant signaling pathways. The functional changes associated with EMT were investigated using the scratch assay, Transwell migration assay, and collagen gel contraction assay. CCK-8 was used to determine the cell viability of phRPE cells. RESULTS: On day 7 and 14 after laser induction in mice, intravitreal injection of luteolin dramatically decreased the immunolabeled sizes of both collagen I and IB4, as well as the amount of colocalized double immunostaining of α-SMA and RPE65 in laser-induced SF lesions. In vitro, TGFß1-treated phRPE cells demonstrated increased cell migration and contraction capacity, accompanied with considerable overexpression of fibronectin, α-SMA, N-cadherin and vimentin, as well as downregulation of E-cadherin and ZO-1. The above changes were largely inhibited by luteolin co-incubation. Mechanistically, luteolin could evidently decrease the phosphorylation of Smad2/3, whereas increase the phosphorylation of YAP in TGFß1-treated phRPE cells. CONCLUSION: This study demonstrates that luteolin exhibits the anti-fibrotic effect in a laser-induced mouse model by inhibiting EMT of RPE cells via deactivating Smad2/3 and YAP signaling, which provides a potential natural compound for the prevention and treatment of SF and fibrosis-related diseases.

Salvianolic Acid A Protects against Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting Neutrophil NETosis.

Salvianolic acid A (SAA) is one of bioactive polyphenol extracted from a Salvia miltiorrhiza (Danshen), which was widely used to treat cardiovascular disease in traditional Chinese medicine. SAA has been reported to be protective in cardiovascular disease and ischemia injury, with anti-inflammatory and antioxidative effect, but its role in acute lung injury (ALI) is still unknown. In this study, we sought to investigate the therapeutic effects of SAA in a murine model of lipopolysaccharide- (LPS-) induced ALI. The optimal dose of SAA was determined by comparing the attenuation of lung injury score after administration of SAA at three different doses (low, 5 mg/kg; medium, 10 mg/kg; and, high 15 mg/kg). Dexamethasone (DEX) was used as a positive control for SAA. Here, we showed that the therapeutic effect of SAA (10 mg/kg) against LPS-induced pathologic injury in the lungs was comparable to DEX. SAA and DEX attenuated the increased W/D ratio and the protein level, counts of total cells and neutrophils, and cytokine levels in the BALF of ALI mice similarly. The oxidative stress was also relieved by SAA and DEX according to the superoxide dismutase and malondialdehyde. NET level in the lungs was elevated in the injured lung while SAA and DEX reduced it significantly. LPS induced phosphorylation of Src, Raf, MEK, and ERK in the lungs, which was inhibited by SAA and DEX. NET level and phosphorylation level of Src/Raf/MEK/ERK pathway in the neutrophils from acute respiratory distress syndrome (ARDS) patients were also inhibited by SAA and DEX in vitro, but the YEEI peptide reversed the protective effect of SAA completely. The inhibition of NET release by SAA was also reversed by YEEI peptide in LPS-challenged neutrophils from healthy volunteers. Our data demonstrated that SAA ameliorated ALI via attenuating inflammation, oxidative stress, and neutrophil NETosis. The mechanism of such protective effect might involve the inhibition of Src activation.

Quantitative Evaluation of Chronic Obstructive Pulmonary Disease and Risk Prediction of Acute Exacerbation by High-Resolution Computed Tomography.

Objective: It is imperative to popularize the tertiary prevention of chronic obstructive pulmonary disease (COPD) and to improve the diagnosis and treatment. Methods: COPD patients were divided into mild (n = 18), moderate (n = 20), severe (n = 24), and extremely severe (n = 22) groups for performing high-resolution computed tomography (HRCT) and pulmonary function test. Serum procalcitonin (PCT) and high-sensitivity C-reactive protein (hs-CRP) were detected, and the occurrence rate of acute exacerbation COPD (AECOPD) was recorded during a 12-months follow-up period. Results: With an increase in the severity grade, the HRCT indexes, including emphysema index (EI), 1st and 15th percentile of inspiratory attenuation distribution (Perc1 and Perc15), ratio of expiratory/inspiratory mean lung density (MLDex/in) and lung volume (LVex/in), and ratio of the wall thickness to the outer diameter of the lumen (TDR), as well as percentage of the wall area to the total cross-sectional area (WA%) were increased with a decreased change in relative lung volume with attenuation values between -860 and -950 HU (RVC-860to -950) and lumen area (A i). These were correlated with the ratio of forced expiratory volume in 1 sec (FEV1) over forced vital capacity (FVC) (FEV1/FVC), the percentage of FEV1 the predicted value (FEV1%), and ratio of residual volume to total lung volume (RV/TLC). Body mass index, MLDex/in, FEV1%, FEV1/FVC, and PCT had a predictive value to AECOPD, with the combined AUC of 0.812. Conclusions: HRCT imaging effectively classifies the severity of COPD, which combined with BMI, PFT, and serum PCT can predict the risk of AECOPD.

Characterization and genomic analysis of an efficient dibutyl phthalate degrading bacterium Microbacterium sp. USTB-Y.

A promising bacterial strain for biodegrading dibutyl phthalate (DBP) was successfully isolated from activated sludge and characterized as a potential novel Microbacterium sp. USTB-Y based on 16S rRNA sequence analysis and whole genome average nucleotide identity (ANI). Initial DBP of 50 mg/L could be completely biodegraded by USTB-Y both in mineral salt medium and in DBP artificially contaminated soil within 12 h at the optimal culture conditions of pH 7.5 and 30 â„ƒ, which indicates that USTB-Y has a strong ability in DBP biodegradation. Phthalic acid (PA) was identified as the end-product of DBP biodegraded by USTB-Y using GC/MS. The draft genome of USTB-Y was sequenced by Illumina NovaSeq and 29 and 188 genes encoding for putative esterase/carboxylesterase and hydrolase/alpha/beta hydrolase were annotated based on NR (non redundant protein sequence database) analysis, respectively. Gene3781 and gene3780 from strain USTB-Y showed 100% identity with dpeH and mpeH from Microbacterium sp. PAE-1. But no phthalate catabolic gene (pht) cluster was found in the genome of strain USTB-Y. The results in the present study are valuable for obtaining a more holistic understanding on diverse genetic mechanisms of PAEs biodegrading Microbacterium sp. strains.

Genetic and Functional Evaluation of the Role of FOXO1 in Antituberculosis Drug-Induced Hepatotoxicity.

BACKGROUND: The accumulation of the hepatotoxic substance protoporphyrin IX (PPIX) induced by aminolevulinate synthase 1 (ALAS1) activation is one of the important mechanisms of antituberculosis drug-induced hepatotoxicity (ATDH). Forkhead box protein O1 (FOXO1) may activate ALAS1 transcription. However, little is known about their roles in ATDH; we performed a study to determine the association between polymorphisms in the two genes and ATDH susceptibility. Then, we verified this possible association by cellular functional experiments. MATERIALS AND METHODS: Tag single-nucleotide polymorphisms (TagSNPs) in the two genes were genotyped in 746 tuberculosis patients. The frequencies of the alleles, genotypes, genetic models, and haplotype distribution of the variants were compared between the case and control groups. L-02 cells and HepG2 cells were incubated with the indicated concentration of isoniazid (INH) and rifampicin (RIF) for the desired times, and then the expression levels of ALAS1 and FOXO1 mRNAs and proteins were detected. HepG2 cells were transiently transfected with FOXO1 siRNA to observe the effect of changes in the FOXO1 expression on the cell survival rate and ALAS1 expression. RESULTS: The C allele at rs2755237 and the T allele at rs4435111 in the FOXO1 gene were associated with a decreased risk of ATDH. The expression of ALAS1 in both L-02 cells and HepG2 cells was increased by the coadministration of INH/RIF (600/200 µM) for 24 h. Although FOXO1 expression was reduced slightly by the same treatment, its content in the nucleus was significantly increased. However, the cell survival rate and ALAS1 expression level were not significantly altered by the downregulation of FOXO1 in HepG2 cells. CONCLUSIONS: Variants of the rs4435111 and rs2755237 loci in the FOXO1 gene were associated with susceptibility to ATDH. Coadministration of INH/RIF promoted the transfer of FOXO1 from the cytoplasm to the nucleus, but the functional significance of its nuclear translocation requires further verification.

Phytoremediation of soil heavy metals (Cd and Zn) by castor seedlings: Tolerance, accumulation and subcellular distribution.

Cd and Zn pollution was observed to often occur simultaneously in soils. However, previous studies focused on single heavy metal instead of Cd and Zn combined pollution. Castor (Ricinus communis) is considered to have great potential for contaminated soil remediation. The resistance of castor seedlings to heavy metals and the mechanism behind it remain unknown. In this study, the tolerance and accumulation ability of castor seedlings to Cd and Zn were investigated, and the accumulation mechanism involving the subcellular distribution in different tissues was further explored. The results on biomass and chlorophyll revealed that castor seedlings have good tolerance to the pollution with 0-5 mg/kg Cd and 380 mg/kg Zn, while not to the heavy pollution with 25 mg/kg Cd and 380 mg/kg Zn. The maximum accumulation concentrations of Cd and Zn, 175.3 mg Cd/kg and 386.8 mg/kg Zn, appeared in castor seedling root instead of stem and leaf, indicating that root played a significant part in accumulating Zn and Cd. The relative low dosage of Cd (0-5 mg/kg) promoted the accumulation of Zn in the subcellular component, while high dosage (25 mg/kg) inhibited the accumulation of Zn. In subcellular accumulation and distribution of castor seedlings, Cd (27.1%-69.4%) and Zn (39.6%-66.6%) in the cell wall was the highest. With the increase of Cd addition, the accumulation of Cd increased in cell wall while decreased in organelle and soluble fraction. Hydroxyl, amino, amides and carboxyl functional groups on cell wall might provided the main binding sites for Cd and Zn.

The Variant at TGFBRAP1 but Not TGFBR2 Is Associated with Antituberculosis Drug-Induced Liver Injury.

BACKGROUND: TGFBRAP1 and TGFBR2 play important roles in the TGF-ß/smad signalling pathway and may disturb liver homeostasis by regulating liver injury and renewal. However, little is known about the association between their genetic polymorphisms and antituberculosis drug-induced liver injury (ATDILI), so we explored the association between their variants and the susceptibility to ATDILI. MATERIALS AND METHODS: A total of 746 tuberculosis patients were prospectively enrolled, and fifteen selected SNPs were genotyped. The allele, genotype, and genetic model frequencies of the variants were compared between patients with or without ATDILI, as well as the joint effect analysis of SNP-SNP interactions. The odds ratio (OR) with the corresponding 95% confidence interval (CI) was calculated. RESULTS: The A variant at rs17687727 was significantly associated with an increased risk for ATDILI (OR 1.55; 95% CI: 1.08-2.22; p = 0.016), which is consistent with the results in the additive and dominant models. Other allele, genotype, and genetic model frequencies were similar in the two groups for the other fourteen SNPs (all p > 0.05). CONCLUSION: Our study first implied that the A variant of rs17687727 in TGFBRAP1 influenced the susceptibility to ATDILI in first-line antituberculosis combination treatment in the Han Chinese population in a dependent manner.

Preventive effect of L-carnitine on the disorder of lipid metabolism and circadian clock of mice subjected to chronic jet-lag.

Circadian clock plays an essential role in orchestrating daily physiology, and its disruption can evoke metabolic diseases such as obesity. L-Carnitine can reduce blood lipid levels, and ameliorate fatty liver through regulating lipid metabolism. However, whether L-Carnitine administration may affect the disturbance of lipid metabolism and circadian rhythm of mice induced by prolonged circadian disruption is still unknown. Herein, we investigated the effects of L-Carnitine on conditions of circadian clock and lipid metabolism through a chronic jet-lag mice model which was developed by reversing 12 h light/12 h dark cycle every 4 days for a continuous 12 weeks. Results showed that L-Carnitine administration significantly decreased levels of serum glutamic-oxaloacetic transaminase (GOT) and triglycerides (TG), which were remarkably elevated by chronic jet-lag. More importantly, quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that L-Carnitine supplementation would effectively counteract the negative alterations in gene expression which related to lipid metabolism (Srebp1, Acaca, Fasn, and Scd1), metabolic regulator (mTOR) and circadian rhythm (Bmal1, Per1, Cry1 and Dec1) in the liver of mice subjected to the chronic jet-lag. As a conclusion, L-Carnitine was partly effective in preventing the disruption of circadian clock and lipid metabolic disorders induced by the chronic jet-lag.

A comparison of the biological effects of 125I seeds continuous low-dose-rate radiation and 60Co high-dose-rate gamma radiation on non-small cell lung cancer cells.

OBJECTIVES: To compare the biological effects of 125I seeds continuous low-dose-rate (CLDR) radiation and 60Co γ-ray high-dose-rate (HDR) radiation on non-small cell lung cancer (NSCLC) cells. MATERIALS AND METHODS: A549, H1299 and BEAS-2B cells were exposed to 125I seeds CLDR radiation or 60Co γ-ray HDR radiation. The survival fraction was determined using a colony-forming assay. The cell cycle progression and apoptosis were detected by flow cytometry (FCM). The expression of the apoptosis-related proteins caspase-3, cleaved-caspase-3, PARP, cleaved-PARP, BAX and Bcl-2 were detected by western blot assay. RESULTS: After irradiation with 125I seeds CLDR radiation, there was a lower survival fraction, more pronounced cell cycle arrest (G1 arrest and G2/M arrest in A549 and H1299 cells, respectively) and a higher apoptotic ratio for A549 and H1299 cells than after 60Co γ-ray HDR radiation. Moreover, western blot assays revealed that 125I seeds CLDR radiation remarkably up-regulated the expression of Bax, cleaved-caspase-3 and cleaved-PARP proteins and down-regulated the expression of Bcl-2 proteins in A549 and H1299 cells compared with 60Co γ-ray HDR radiation. However, there was little change in the apoptotic ratio and expression of apoptosis-related proteins in normal BEAS-2B cells receiving the same treatment. CONCLUSIONS: 125I seeds CLDR radiation led to remarkable growth inhibition of A549 and H1299 cells compared with 60Co HDR γ-ray radiation; A549 cells were the most sensitive to radiation, followed by H1299 cells. In contrast, normal BEAS-2B cells were relatively radio-resistant. The imbalance of the Bcl-2/Bax ratio and the activation of caspase-3 and PARP proteins might play a key role in the anti-proliferative effects induced by 125I seeds CLDR radiation, although other possibilities have not been excluded and will be investigated in future studies.

Successful management of chylous ascites with total parenteral nutrition and octreotide in children.

PURPOSE: To evaluate the effects of total parenteral nutrition and octreotide on pediatric patients with chylous ascites post-operative. METHODS: Four patients were diagnosed with chylous ascites from Nov 2009 to Nov 2012. Total parenteral nutrition and octreotide was administered to 2 patients, while the other two only received fasting and total parenteral nutrition. All patients had persistent peritoneal drainage, with the quantity and quality of drainage fluid observed daily. RESULTS: Two patients who received somatostatin therapy completely recovered within 7d without any recurrence while on a normal diet. The other two patients who only received fasting and total parenteral nutrition was cured 24-30 d after therapy. CONCLUSION: Total parenteral nutrition along with octreotide can relieve the symptoms and close the chyle leakage in patients with chylous ascites rapidly. It seems to be an effective therapy available for the treatment of chylous ascites.

Purpose: To evaluate the effects of total parenteral nutrition and octreotide on pediatric patients with chylous ascites post-operative. Methods: Four patients were diagnosed with chylous ascites from Nov 2009 to Nov 2012. Total parenteral nutrition and octreotide was administered to 2 patients, while the other two only received fasting and total parenteral nutrition. All patients had persistent peritoneal drainage, with the quantity and quality of drainage fluid observed daily. Results: Two patients who received somatostatin therapy completely recovered within 7d without any recurrence while on a normal diet. The other two patients who only received fasting and total parenteral nutrition was cured 24-30d after therapy. Conclusion: Total parenteral nutrition along with octreotide can relieve the symptoms and close the chyle leakage in patients with chylous ascites rapidly. It seems to be an effective therapy available for the treatment of chylous ascites.

[Start-up of high solid anaerobic digestion process for treating straw with sludge cake as nitrogen additive].

Straw co-digestion with sludge cake as nitrogen additive from wastewater treatment plant (WWTP) can solve the pollution problems of improper treatment of straw and sludge. Moreover the products, including biogas and organic manures, can relieve the problems of energy supply and soil infertility in countryside. The properties of the start-up periods of two anaerobic digestors were studied. The result showed that the efficiency of the anaerobic digestors rose after pre-treating the straw by NaOH and adjusting C/N by adding 5% sludge. The start-up period lasted 930 h. The anaerobic bacteria activity has recovered and was acclimated in the start-up period. The solid content of the feeding was 12%-16% and the biogas-production rate was 0.15-0.18 L/h at the end of start-up period. The total solid and volatile solid degradation rates were 54% and 65% in the 1st reactor, and 67% and 75% in the 2nd reactor, respectively. During the start-up period, COD concentration and NH4+ -N concentration were rather low, which were 1000-6000 mg/L and 200-600 mg/L, respectively.

[Heavy metal stabilization in municipal solid waste incineration fly ash using soluble phosphate].

The physico-chemical characteristics of municipal solid waste incineration fly ashes was analyzed. It indicated that the main elements of fly ashes are Ca, Cl, K, S and Si, and many heavy metals such as Pb, Zn, Cu, Mn and Cr can be found in fly ashes, and the heavy metal leaching toxicity such as Pb, Cu, Zn and Cd is much higher than the standards. A new kind of stabilization agent-soluble phosphate is chosen to treat with fly ash, different influence factors including agent dosage, curing time and pH are taken into consideration to test its stabilization for the stabilized fly ash. The experimental results indicate that fly ash treated with soluble phosphate can have excellent stabilization effect, and the leaching toxicity for Pb, Cd and Zn can be reduced by 97.5%, 91.6% and 95.5% at a phosphate dosage of 3%; curing time can not influence its stabilization; and, stabilized fly ash using soluble phosphate can keep long-term stabilization at a broad pH value.