Anti-inflammatory and anti-apoptotic effects of N-acetylcysteine in diabetic rat corneal epithelium

AIM: To characterize the anti-inflammatory and anti-apoptotic effects of N-acetylcysteine (NAC) in streptozotocin (STZ)-induced diabetic rat corneal epithelium and human corneal epithelial cells (HCECs) exposed to a high-glucose environment. METHODS: HCECs were incubated in 0, 5, 50 mmol/L glucose medium, or 50 mmol/L glucose medium with NAC for 24h. Diabetes was induced in rats by intraperitoneal injection of 65 mg/kg STZ and some of these rats were topically administered NAC to corneas with 3 mice per group. We characterized receptor for advanced glycation end-products (RAGE) expression using immunofluorescence, and interleukin (IL)-1β and cleaved caspase-3 (CCAP-3) expression using immunohistochemistry. Circulating tumor necrosis factor (TNF)-α concentration was measured by ELISA and cleaved poly-ADP ribose polymerase (PARP) concentration was quantified by Western blotting. Apoptotic cells were detected using TUNEL assay and annexin V and propidium iodide staining. RESULTS: Diabetic rats had higher expression of RAGE (2.46±0.13 fold), IL-1β, and CCAP-3 in apoptotic cells of their corneas than control rats. The expression of RAGE (1.83±0.11 fold), IL-1β, and CCAP-3, and the number of apoptotic cells, were reduced by topical NAC treatment. HCECs incubated in 50 mmol/L glucose medium showed high concentrations of TNF-α (310±2.00 pg/mL) and cleaved PARP (7.43±0.56 fold), and more extensive apoptosis than cells in 50 mmol/L glucose medium. However, the addition of NAC reduced the concentrations of TNF-α (153.67±2.31 pg/mL) and cleaved PARP (5.55±0.31 fold) and the number of apoptotic cells. CONCLUSION: NAC inhibits inflammation and apoptosis in the corneas of diabetic rats and HCECs maintained in a high-glucose environment.

Lactic Acid Transport Mediated by Aquaporin-9: Implications on the Pathophysiology of Preeclampsia

Aquaporin-9 (AQP9) expression is significantly increased in preeclamptic placentas. Since feto-maternal water transfer is not altered in preeclampsia, the main role of AQP9 in human placenta is unclear. Given that AQP9 is also a metabolite channel, we aimed to evaluate the participation of AQP9 in lactate transfer across the human placenta. Explants from normal term placentas were cultured in low glucose medium with or without L-lactic acid and in the presence and absence of AQP9 blockers (0.3 mM HgCl2 or 0.5 mM Phloretin). Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase release. Apoptotic indexes were analyzed by Bax/Bcl-2 ratio and Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick-End Labeling assay. Heavy/large and light/small mitochondrial subpopulations were obtained by differential centrifugation, and AQP9 expression was detected by Western blot. We found that apoptosis was induced when placental explants were cultured in low glucose medium while the addition of L-lactic acid prevented cell death. In this condition, AQP9 blocking increased the apoptotic indexes. We also confirmed the presence of two mitochondrial subpopulations which exhibit different morphologic and metabolic states. Western blot revealed AQP9 expression only in the heavy/large mitochondrial subpopulation. This is the first report that shows that AQP9 is expressed in the heavy/large mitochondrial subpopulation of trophoblasts. Thus, AQP9 may mediate not only the lactic acid entrance into the cytosol but also into the mitochondria. Consequently, its lack of functionality in preeclamptic placentas may impair lactic acid utilization by the placenta, adversely affecting the survival of the trophoblast cells and enhancing the systemic endothelial dysfunction.

Construction of an Artificial Biosynthetic Pathway for the Styrylpyrone Compound 11-Methoxy-Bisnoryangonin Produced in Engineered Escherichia coli

A cDNA clone (named pnpks), which shows high homology to the known chalcone synthase (CHS)-like type III PKS, was obtained from the leaves of Piper nigrum. The PnPKS protein with ferulic acid catalyzed lactonization instead of chalcone or stilbene formation. The new product was characterized as a styrylpyrone, 11-methoxy-bisnoryangonin, which is the lactonization compound of a linear triketide formed as the reaction product of PnPKS protein with ferulic acid. These results show that pnpks encodes a styrylpyrone synthase (SPS)-like PKS that catalyzes two-chain elongation with feruloyl CoA-linked starter substrates. Although these styrylpyrone compounds are promising for use in human healthcare, they are mainly obtained by extraction from raw plant or mushroom sources. For de novo synthesis of 11-methoxy-bisnoryangonin in the heterologous host Escherichia coli from a simple sugar as a starter, the artificial biosynthetic pathway contained five genes: optal, sam5, com, and 4cl2nt, along with the pnpks gene. The engineered L-tyrosine overproducing E. coli ∆COS1 strain, in which five biosynthetic genes were cloned into two vectors, pET-opT5M and pET22-4P, was cultured for 24 h in a minimal glucose medium containing ampicillin and kanamycin. As a result, 11-methoxy-bisnoryangonin production of up to 52.8 mg/L was achieved, which is approximately 8.5-fold higher than that in the parental E. coli strain harboring a plasmid for 11-methoxy-bisnoryangonin biosynthesis. As a potential styrylpyrone compound, 11-methoxy-bisnoryangonin, was successfully produced in E. coli from a simple glucose medium, and its production titer was also increased using engineered strains. This study provides a useful reference for establishing the biological manufacture of styrylpyrone compounds.

Excess Glucose Impedes the Proliferation of Skeletal Muscle Satellite Cells Under Adherent Culture Conditions

Glucose is a major energy source consumed by proliferating mammalian cells. Therefore, in general, proliferating cells have the preference of high glucose contents in extracellular environment. Here, we showed that high glucose concentrations impede the proliferation of satellite cells, which are muscle-specific stem cells, under adherent culture conditions. We found that the proliferation activity of satellite cells was higher in glucose-free DMEM growth medium (low-glucose medium with a glucose concentration of 2 mM) than in standard glucose DMEM (high-glucose medium with a glucose concentration of 19 mM). Satellite cells cultured in the high-glucose medium showed a decreased population of reserve cells, identified by staining for Pax7 expression, suggesting that glucose concentration affects cell fate determination. In conclusion, glucose is a factor that decides the cell fate of skeletal muscle-specific stem cells. Due to this unique feature of satellite cells, hyperglycemia may negatively affect the regenerative capability of skeletal muscle myofibers and thus facilitate sarcopenia.

CircRNA-DAPK1 promotes vascular cell pyroptosis in diabetes by regulating the circ-DAPK1/miR-4454/TXNIP axis.

Background Circular RNAs have been demonstrated to play an important role in the development of vascular diseases. However, little is known about the role of circ-021774, also named circ-DAPK1, in vascular cell pyroptosis. Methods Circ-DAPK1 was selected from circular RNA sequencing data of HUVECs treated with high glucose medium and normal medium. RT-qPCR was used to determine the expression of circ-DAPK1 in vivo and in vitro. Dual luciferase reporter assay, fluorescence in situ hybridization (FISH) and RNA immunoprecipitation (RIP) were performed to prove the interaction of circ-DAPK1, miRNA-4454 and thioredoxin-interactingprotein (TXNIP). Adeno-associated virus (AAV) was injected intravenously to establish mouse models. PI staining, western-blot and transmission electron microscopy (TEM) analyses were performed to identify the role of circ-DAPK1 in promoting pyroptosis. Results We found that circ-DAPK1 was highly expressed in high glucose medium cultured HUVECs and db/db mice. In vitro and in vivo experiments demonstrated that circ-DAPK1 knockdown decreased the number of PI+ cells, the expression of ASC, NLRP3, GSDMD-N, cleaved caspase-1, IL-18 and IL-1β. In a mechanistic study, the circ-DAPK1/miRNA-4454/TXNIP signaling axis was demonstrated to promote vascular cell pyroptosis in diabetes. Conclusions Circ-DAPK1 functions as a promoter of vascular cell pyroptosis in diabetes via the circ-DAPK1/miRNA-4454/TXNIP signaling axis.

Evaluation of Cells and Medium Optimization for Invitro Model of Diabetic and Electrolyte Imbalance

Metabolism syndrome has many negative impacts on human health. Various efforts and methods are attempted in the treatment of this disease. One of the methods used is CRISPR/Cas9 gene therapy. Re-testing of knock out cells using the CRISPR/Cas9 method is needed to evaluate its success. In conducting the test, the right medium is needed so that the results are optimal and can be evaluated properly. In this study, we optimized the medium for three types of cells (fibroblasts, myoblasts and macrophages) in high and low glucose medium to evaluate gene knockout results. The medium was modified by adding high concentrations of glucose and sodium. The results, in macrophage culture, giving variations in glucose concentration in low glucose medium gave a significantly different percentage of live cells between treatments, while the treatment with variations in glucose concentration in macrophages in high glucose medium and fibroblasts and myoblasts in high and low glucose medium did not show any difference in the percentage of living cells. In the treatment of various concentrations of natrium, macrophages, fibroblasts and myoblasts on high and low glucose medium all showed significantly different percentages of living cells. Therefore, DMEM low glucose medium is suitable as a medium for the treatment of high glucose and natrium induction in macrophage cells, but is not suitable for fibroblast and myoblast cells.

Glutamate dehydrogenase 1 mediated glutaminolysis sustains HCC cells proliferation and survival under glucose deprivation

Background: It is generally believed that tumor cells could sustain its proliferation and survival under different nutrient status according to a so-called metabolic flexibility. How the metabolic flexibility of glutamine metabolism of HCC cells behaves under different glucose conditions has not yet been fully elucidated. In this study, we investigated how the glutamine metabolism modulate the proliferation and survival of HCC cells in response to different glucose conditions and explored the underlying molecular mechanism.Methods: Two cell lines SK-Hep-1 and PLC/PRF/5 were used to evaluate the glutamine addiction of HCC cells. Then, the cells were cultivated in high glucose medium (25mM glucose) and low glucose medium (1.0 mM glucose), respectively, to investigate whether glutaminolysis changed in response to different glucose levels. And, the underlying mechanism of glutamate dehydrogenase 1 (GDH1) sustaining HCC cells survival under glucose deprivation was explored. Additionally, the underlying correlation of GDH1 and glutamate–oxaloacetate transaminase 1 (GOT1) in glucose -poor HCC tissue was investigated.Results: HCC cells were addicted to glutamine. The glutaminolysis of HCC cells was different in response to different glucose conditions. That is, glutamate transaminases GOT1 involved glutamine metabolism played a dominant role in regulating cell growth when glucose was sufficient, while deaminase GDH1 mediated glutaminolysis became dominant when glucose was limited. Mechanically, low-glucose treated HCC cells could induce an elevated expression of GDH1 to supplement the TCA cycles in respond to glucose deprivation. Additionally, we further uncovered an underlying negative association between GDH1 and GOT1 in HCC tissues with decreased glucose levelsConclusions: GDH1 mediated pathway played a leading role in maintaining cell proliferation and survival under low glucose condition. By contrast, GOT1 mediated pathway was activated under high glucose condition. Mechanically, highly expressed GDH1 could drive the TCA cycle in response to glucose deprivation. Besides, there was a potential negative correlation between GDH1 and GOT1 in glucose-poor HCC tissues.

The Role of PPARγ in Hyperglycemia-induced Deleterious Effect on Chondrocytes

Aims: There is a well-established link between OA and diabetes, and study have shown that hyperglycemia might play an important role in the occurrence and development of OA. Accumulative evidence suggested that PPARγ was involved in AGEs-related disease, including diabetes and OA. The study was designed to investigate the effects of hyperglycemia on the expression of PPARγ in chondrocytes and whether PPARγ agonist pioglitazone had a chondroprotective effect.Main methods: Primary human chondrocytes were incubated with different concentration of glucose medium(5.5mM-30mM) in the presence or absence of PPARγ agonist pioglitazone. The AGEs formation level in chondrocytes culture medium was detected by AGEs specific ELISA kits. The expression of IL-1, MMP-13, TNF-α, PPARγ was determined by western blotting and real-time PCR.Key findings: The AGEs fomation level was time-dependently and dose-dependently increased in chondrocyte culture media. Hyperglycemia could enhance the expression of IL-1β, TNF-α, MMP-13, but the level of PPARγ was decreased in a time-dependent and dose-dependent manner, which was inhibited by PPARγ agonist pioglitazone. Noteworthy, the maximum effect was found to at 20mM glucose medium for 24h.Significance: Hyperglycemia could increase the AGEs formation level and induce inflammatory response and matrix degradation reaction in chondrocytes. PPARγ agonists pioglitazone had a chondroprotective effect via inhibit inflammatory response and matrix degradation reaction. PPARγ could be a potential target for pharmacologic intervention in the treatment of diabetic-induced OA.

Dapagliflozin activates AMPK to attenuate cardiac dysfunction and oxidative stress under hypoxia/reoxygenation-caused damage

Background: Emerging evidence demonstrated Dapagliflozin (DAPA), an inhibitor of type II sodium-glucose cotransporter-2, prevented various cardiovascular events. However, the detailed mechanisms underlying its cardioprotective properties remained largely unknown. In the present study, we sought to investigate the effects of DAPA on the cardiac ischemia/reperfusion (I/R) injury and study the mechanisms of DAPA-provided cardioprotection.Methods: For in intro studies, cardiac myoblast H9c2 cells were exposed to hypoxia with no-glucose medium for 1 hr than followed a reoxygenation with high-glucose medium for 4 hr. DAPA was treated before hypoxia/reoxygenation (H/R) exposure. For in vivo investigations, I/R was instigated in Sprague-Dawley (SD) rats using ligation of the left anterior descending coronary artery (LAD). DAPA was given daily by gavage for 5 days before I/R induction.Results: Results from in vitro experiments showed that DAPA induced the phosphorylation of adenosine 5'-monophosphate activated protein kinase (AMPK), resulting in the downregulation of phosphorylated protein kinase C (PKC) in the cardiac myoblast H9c2 cells following H/R condition. We demonstrated that DAPA treatment diminished the H/R-elicited oxidative stress via the AMPK/ PKC/ NADPH oxidase (Nox) pathway. In addition, DAPA prevented the H/R-induced abnormality of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) expression, mitochondrial membrane potential, and mitochondrial DNA copy number through AMPK/ PKC/ Nox signaling. Besides, DAPA reversed the apoptosis-associated changes, including H/R-suppressed Bcl-2 and H/R-induced expression of phosphorylated p53, Bax cytochrome c, and activated caspase 3 via AMPK/ PKC/ Nox/ PGC-1α signaling. Furthermore, we demonstrated that DAPA improved the I/R-induced cardiac dysfunction by echocardiography and abrogated the I/R-elicited apoptotic cells by terminal deoxynucleotidyl transferase dUTP nick end labeling assay in the myocardium of rats. Also, the administration of DAPA mitigated the production of two myocardial infarction markers, creatine phosphokinase isoenzymes and lactate dehydrogenase.Conclusion: In conclusion, our data suggested that DAPA treatment holds the potential to ameliorate the I/R-elicited oxidative stress and the following cardiac apoptosis via AMPK/ PKC/ Nox/ PGC-1α signaling, which attenuates the cardiac dysfunction caused by I/R injury.