Apoptotic and non-apoptotic roles of caspases in placenta physiology and pathology.

Caspases, a family of cysteine proteases, are pivotal regulators of apoptosis, the tightly controlled cell death process crucial for eliminating excessive or unnecessary cells during development, including placental development. Collecting research has unveiled the multifaceted roles of caspases in the placenta, extending beyond apoptosis. Apart from their involvement in placental tissue remodeling via apoptosis, caspases actively participate in essential regulatory processes, such as trophoblast fusion and differentiation, significantly influencing placental growth and functionality. In addition, growing evidence indicates an elevation in caspase activity under pathological conditions like pre-eclampsia (PE) and intrauterine growth restriction (IUGR), leading to excessive cell death as well as inflammation. Drawing from advancements in caspase research and placental development under both normal and abnormal conditions, we examine the significance of caspases in both cell death (apoptosis) and non-cell death-related processes within the placenta. We also discuss potential therapeutics targeting caspase-related pathways for placenta disorders.

Standardised Ethanol Extract of Tithonia diversifolia (Hemsley) A Gray Leaves Improve Insulin Sensitivity and Increase Mitochondrial DNA Copy Numbers in Skeletal Muscles of Streptozotocin-Nicotinamide-Induced Rats.

Background: In this study, we examined the anti-diabetic activity of standardised extracts of Tithonia diversifolia (Hemsley) A Gray (T. diversifolia) leaves for their effects on insulin resistance and mitochondrial DNA (mtDNA) copy number. Methods: T. diversifolia leaves were extracted using an ultrasound-assisted method and standardised using Tagitinin C. There were six groups: i) normal control; ii) diabetic group; iii) metformin group (300 mg/kg) and iv) groups treated with three different doses of extract (50 mg/kg, 100 mg/kg and 150 mg/kg). Blood samples were taken before and after 28 days of treatment for fasting plasma glucose (FPG) and insulin analysis, which were used for a Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) calculation. The soleus and gastrocnemius muscles were harvested after 28 days of treatment for the measurement of mtDNA copy number. Results: The results showed an improvement in blood glucose levels and HOMA-IR scores in all treatment groups. The results of mtDNA copy number analysis also revealed significant improvement with the highest number observed at an extract dose of 100 mg/kg in which the mtDNA copy number increased up to 3 times in the soleus muscles (P < 0.001). Conclusion: T. diversifolia extract has the potential to be used as an anti-diabetic agent that improves insulin resistance, possibly by increasing mtDNA content.

The Effect of PRKAA2 Variation on Type 2 Diabetes Mellitus in the Asian Population: A Systematic Review and Meta-Analysis.

The prevalence of type 2 diabetes mellitus (T2DM) is increasing among Asians. The adenosine monophosphate-activated protein kinase (AMPK) increases T2DM risk through insulin resistance. Glucose levels are related to AMPK subunit α2 encoded by PRKAA2. This systematic review and meta-analysis aimed to analyse the association between PRKAA2 variation and T2DM risk. Publication search related to PRKAA2 and T2DM used PubMed, ProQuest, and ScienceDirect databases. Article selection based on inclusion and exclusion criteria only included Japanese and Chinese populations. This meta-analysis used five genotype models to estimate the effect of PRKAA2 variation and T2DM risk. Additionally, a fixed-effect model was selected to measure the pooled size effect if P > 0.05 or I2 < 50%. Qualitative analysis included four eligible studies, and meta-analysis included only two studies because both showed data concerning rs2746342 variation. Patients with G allele are 1.45 times more likely to have T2DM than patients with T allele (95% confidence interval [CI]: 1.20, 1.76; P: 0.0001). Notably, patients who had GG genotype have 1.96 times higher risk of T2DM compared with those with TT genotype (95% CI: 1.34, 2.87; P: 0.0005), dominant model (odds ratio [OR]: 1.75; 95% CI: 1.32, 2.31; P: 0.001), and recessive model (OR: 1.43; 95% CI: 1.01, 2.01; P: 0.04). PRKAA2 variation, especially in rs2746342, has an association with T2DM risk in the G allele, additive, dominant, and recessive models. G allele might be the most contributable factor in increasing T2DM susceptibility.

The effect of Kappaphycus alvarezii active fraction on oxidative stress and inflammation in streptozotocin and nicotinamide-induced diabetic rats.

BACKGROUND: High glucose concentration increases the glycation process which leads to oxidative stress and inflammation, that can cause complications in diabetes. Several medicinal plants have been used in the treatment of diabetes and its complications. One of them is Kappaphycus alvarezii, an algae that has known antidiabetic abilities. This study aimed to examine the effect of K. alvarezii active fraction on plasma hydrogen peroxide (H2O2) and Tumor Necrosis Factor α (TNFα) levels, renal NADPH oxidase 4 (NOX4) and Nuclear Factor κ B (NFκB) gene expressions. METHODS: Active fraction was obtained from bioassay-guided fractionation with antiglycation ability. In vivo study was performed on twenty Wistar male rats. The level of H2O2 was measured using H2O2 Assay Kit, the Optical Density value measured using spectrophotometer at a wavelength of 405 nm. Plasma TNFα level was measured using ELISA. Renal NOX4 and NFκB gene expression was analyzed using qPCR. RESULTS: Active fraction significantly reduced plasma H2O2 but not TNFα levels. Furthermore, renal NOX4 gene expression was lower in the diabetic rat group treated with active fraction compared to the untreated group but not NFκB gene expression. CONCLUSIONS: K. alvarezii active fraction has an activity to reduce plasma H2O2 as well as renal NOX4 gene expression. Therefore, this fraction could be developed as a potential candidate for diabetes treatment through oxidative stress mechanisms.

Piper crocatum Ruiz & Pav. ameliorates wound healing through p53, E-cadherin and SOD1 pathways on wounded hyperglycemia fibroblasts.

INTRODUCTION: Piper crocatum Ruiz & Pav (P. crocatum) has been reported to accelerate the diabetic wound healing process empirically. Some studies showed the benefits of P. crocatum in treating various diseases but its mechanisms in diabetic wound healing have never been reported. In the present study we investigated the diabetic wound healing activity of the active fraction of P. crocatum on wounded hyperglycemia fibroblasts (wHFs). METHODS: Bioassay-guided fractionation was performed to get the most active fraction. The selected active fraction was applied to wHFs within 72 h incubation. Mimicking a diabetic condition was done using basal glucose media containing an additional 17 mMol/L D-glucose. A wound was simulated via the scratch assay. The collagen deposition was measured using Picro-Sirius Red and wound closure was measured using scratch wound assay. Underlying mechanisms through p53, αSMA, SOD1 and E-cadherin were measured using western blotting. RESULTS: We reported that FIV is the most active fraction of P. crocatum. We confirmed that FIV \(7.81 µg/ml, 15.62 µg/ml, 31.25 µg/ml, 62.5 µg/ml, and 125 µg/ml) induced the collagen deposition and wound closure of wHFs. Furthermore, FIV treatment (7.81 µg/ml, 15.62 µg/ml, 31.25 µg/ml) down-regulated the protein expression level of p53 and up-regulated the protein expression levels of αSMA, E-cadherin, and SOD1. DISCUSSION/CONCLUSIONS: Our findings suggest that ameliorating collagen deposition and wound closure through protein regulation of p53, αSMA, E-cadherin, and SOD1 are some of the mechanisms by which FIV of P. crocatum is involved in diabetic wound healing therapy.

The effect of Kappaphycus alvarezii fraction on plasma glucose, Advanced Glycation End-products formation, and renal RAGE gene expression.

BACKGROUND: Kappaphycus alvarezii (Doty) Doty ex P.C.Silva is a red algae with antioxidant and antiglycation activities. Algae still have not been widely used for treating diabetes, especially to prevent complications. The purpose of this study was to examine the effect of active fractions from Kappaphycus alvarezii on plasma glucose level, glycation process and renal RAGE gene expression. METHODS: This study used bioassay-guided fractionation, consisting of three stages: extraction, partition, and fractionation. These processes were monitored with Thin Layer Chromatography and the BSA-Glucose method to select the best extract with antiglycation activity (calculated as the percentage of inhibition and IC50). The selected active fraction from four fractions was further used for in vivo study, which was conducted with hyperglycemic Wistar male rats. Plasma glucose level was measured using GOD-PAP methods, while plasma glycated albumin (GA) and Nε- (carboxymethyl) lysine (CML) levels were measured using ELISA. Renal RAGE gene expression was analyzed using qPCR. RESULTS: Fraction II was selected as the active fraction of Kappaphycus alvarezii showing antiglycation activity with the highest percentage of inhibition and the lowest IC50. This fraction significantly reduced plasma GA and CML levels, but it did not significantly reduce plasma glucose level. Furthermore, renal RAGE gene expression was lower in the diabetic rat group treated with this active fraction compared to the untreated group. CONCLUSIONS: This study successfully identified an active fraction of Kappaphycus alvarezii with antiglycation activity to reduce plasma GA and CML levels as well as renal RAGE gene expression. Therefore, this fraction could be developed as a potential candidate for treating diabetes.

PRKAA2 variation and the clinical characteristics of patients newly diagnosed with type 2 diabetes mellitus in Yogyakarta, Indonesia.

Background: Adenosine monophosphate (AMP)-activated protein kinase (AMPK; EC 2.7.11.31) enzymes play a pivotal role in cell metabolism. They are involved in type 2 diabetes mellitus (T2DM) pathogenesis. Genetic variation of PRKAA2 coding for the AMPK α2 catalytic subunit (AMPKα2) is reported to be associated with susceptibility for T2DM. Objectives: To determine the association between PRKAA2 genetic variations (rs2796498, rs9803799, and rs2746342) with clinical characteristics in patients newly diagnosed with T2DM. Methods: We performed a cross-sectional study including 166 T2DM patients from 10 primary health care centers in Yogyakarta, Indonesia. We measured fasting plasma glucose, hemoglobin A1c, serum creatinine, glomerular filtration rate, blood pressure, and body mass index as clinical characteristics. PRKAA2 genetic variations were determined by TaqMan SNP genotyping assay. Hardy-Weinberg equilibrium was calculated using χ2 tests. Results: There was no difference in clinical characteristics for genotypes rs2796498, rs9803799, or rs2746342 (P > 0.05). No significant association was found between PRKAA2 genetic variations and any clinical feature observed. Further subgroup analysis adjusting for age, sex, and waist circumference did not detect any significant association of PRKAA2 genetic variations with clinical characteristics (P > 0.05). Conclusion: PRKAA2 genetic variation is not associated with the clinical characteristics of Indonesian patients with newly diagnosed T2DM.

Centella asiatica (L.) extract attenuates inflammation and improve insulin sensitivity in a coculture of lipopolysaccharide (LPS)-induced 3T3-L1 adipocytes and RAW 264.7 macrophages.

Insulin resistance in obese condition is related to chronic low-grade inflammation which leads to insulin signaling impairment. Centella asiatica (L.) is an herb that exhibits anti-inflammatory and blood sugar-lowering activity (hypoglycemia). The study aims to investigate the molecular mechanism of C. asiatica extract in insulin sensitivity improvement in a coculture of lipopolysaccharide (LPS)-induced 3T3-L1 adipocytes and RAW 264.7 macrophages. A coculture of 3T3-L1 adipocytes and RAW 264.7 macrophages were incubated with LPS to induce insulin resistance in the adipocytes. An extract of C. asiatica was added to coculture cells and after 24 hours, insulin sensitivity and inflammatory response were determined, including glucose consumption, glucose transporter-4 (GLUT-4), insulin receptor substrate-1 (IRS-1), and interleukin-6 (IL-6) mRNA expression. C. asiatica extract at a concentration of 500 µg/mL increased glucose consumption and induced GLUT-4 and IRS-1 mRNA expression significantly in a coculture of LPS-induced 3T3-L1 adipocytes and RAW 264.7 macrophages. The pro-inflammatory cytokines IL-6 mRNA expression was decreased in the coculture cells after treatment with C. asiatica extract at a concentration of 500 µg/mL. This result indicates that C. asiatica has an effect to stimulate glucose consumption in the coculture cells that might be mediated via GLUT-4/IRS-1 pathway as a result of IL-6 inhibition. These findings suggest that the C. asiatica extract inhibits inflammation and improves insulin sensitivity in a coculture of LPS-induced 3T3-L1 adipocytes and RAW 264.7 macrophages.

The effect of 5α-oleandrin on keloid fibroblast activities.

BACKGROUND: Keloids develop due to hyperactivity of keloid fibroblast (KF) in proliferation, migration, and collagen deposition along with low rates of collagen degradation. These are a result of the Wnt/ß catenin signaling pathways under stimulation of TGF-ß. 5α-oleandrin can suppress Wnt-targeted genes of osteosarcoma cells. We aimed to evaluate the anti-fibrotic effects of 5α-oleandrin on KF activities. METHODS: We collected the core of keloid materials from six patients who underwent keloid debulking surgery. Passage 4 of KF cells were then treated with mitomycin-C, 5α-oleandrin, and dilution medium as the negative control. To determine the effective dose of 5α-oleandrin, we diluted 5α-oleandrin into various concentrations. The incubation periods were 24 h, 48 h, and 72 h. The anti-proliferation and anti-fibrotic properties were measured using standard assay. RESULTS: Both the mitomycin-C and 5α-oleandrin treated groups indicated decrease in proliferation index (86.16 ± 4.20% and 73.76 ± 4.94%, respectively), collagen deposition index (90.26 ± 1.72% and 71.35 ± 4.26%, respectively), and migration capacity (33.51 ± 1.50% and 28.57 ± 1.58%, respectively). These were significant changes (p ≤ 0.05) compared to the non-treated group. Antifibrotic activities of 5α-oleandrin in cellular proliferation and collagen deposition were better than mitomycin-C. CONCLUSIONS: The 5α-oleandrin has good antifibrotic effect in keloid fibroblast activities.

Antidiabetic Effect of Rosella-Stevia Tea on Prediabetic Women in Yogyakarta, Indonesia.

BACKGROUND: Rosella tea (Hibiscuss sabdariffa. Linn) with stevia sweetener (Stevia rebaudiana Bertoni) is a combined herbal drink that is expected to have antidiabetic effect by lowering glucose levels in people with diabetes and prediabetes. This research investigates the effect of rosella-stevia tea to decrease fasting blood glucose (FBG) and 2 hours postprandial blood glucose (2-hour postprandial BG) level in prediabetic women. METHOD: This is quasi-experimental research with control and treatment (rosella-stevia tea) group. Each group consists of 12 prediabetic women aged 30-60 years. Rosella-stevia tea (5 g rosella powder, 125 mg stevia sweetener) was administered to the treatment group twice a day for 14 days. RESULT: Rosella-stevia tea consumption affects blood glucose levels. Rosella-stevia tea consumption significantly lowered the FBG level (from 111.25 ± 7.20 mg/dL to 88.58 ± 13.19 mg/dL; p < 0.01) but not the 2-hour postprandial BG level (from 123.25 ± 37.61 mg/dL to 106.92 ± 18.82 mg/dL). There are no significant differences in the control group (FBG level from 106.00 ± 5.27 mg/dL to 102.08 ± 8.36, and 2-hour postprandial BG level from 119.83 ± 16.43 mg/dL to 128.00 ± 23.54 mg/dL). CONCLUSION: Rosella-stevia tea consumption can lower the FBG level but not the 2-hour postprandial BG level in prediabetic women.

The antifibrotic effect of isolate tagitinin C from tithonia diversifolia (Hemsley) A. Gray on keloid fibroblast cell.

INTRODUCTION: Keloids characterized by fibroblast hyperproliferation and depositions of collagen which similar to cancer cells. Tagitinin C is a class of sesquiterpene lactones (SLS) was isolated from the leaves of the moon flower Tithonia diversifolia (Hemsley) A. Gray. The study aim is to evaluate the effects of tagitinin C from Tithonia diversifolia to keloid fibroblasts (KF). METHODS: Monolayer cultures of keloid fibroblast (three passages) were treated with 8 serial concentration of tagitinin C (0.015 to 2) µg/mL during 72 and 120 hours. A positive control using mitomycin C. Cellular viabilities were measured by MTT assay. Collagen depositions were measured by Sirius Red assay for nonsoluble collagen. RESULTS: The reading of the result was conducted by ELISA reader. Data were analyzed by probit regression with SPSS 19 for Windows. The result showed that tagitinin C can inhibit keloid fibroblasts (KF) viability with IC50 0.122 µg/mL (incubation 72h) and 0.039 µg/mL (120h), whereas mitomycin C IC50 0.120 µg/mL (72h) and IC50 of 0.100 µg/mL (120h). At IC50 concentration of tagitinin C on keloid collagen deposition 53.1% (72h) and 44.3% (120h), whereas the IC50 concentration of mitomycin C on keloid collagen deposition 60.4% (72h) and 52.1% (120h). Selectivity index tagitinin C on normal fibroblasts (NF) is 287 for 72h incubation and 791 for 120h incubation. CONCLUSION: It can be concluded that the ability of tagitinin C inhibits KF viability and decreasing keloid collagen deposition is consistent with the concentration (concentration-dependent) and incubation time (time-dependent). Tagitinin C has a low toxicity level on NF with high selectivity index.