Centella asiatica extract ameliorates deoxygenation-induced neurological dysfunction in zebrafish larvae.

Background: Oxygen deprivation (OD) is a critical condition that can lead to brain damage and even death. Current hypoxia management approaches are limited in effectiveness. Centella asiatica (CA), known for its neuroprotective properties, offers a potential alternative for OD treatment. Aims: This study aims to investigate the neuroprotective effects of CA on the expression of brain-derived neurotrophic factor (BDNF) and vesicular glutamate transporter 1 (VGLUT1) in zebrafish larvae under oxygen-deficient conditions. Methods: Zebrafish embryos were subjected to low oxygen levels (1.5 mg/l) 0-2 hours post-fertilization (hpf) until 3 days post-fertilization (dpf), simulating the early stages of OD. Subsequent treatment involved varying concentrations of CA (1.25-5 µg/ml) up to 9 days post-fertilization. The expression levels of BDNF and VGLUT1 were measured using PCR methods. Statistical analysis was conducted using a two-way analysis of variance to evaluate the impact of CA on the expression of BDNF and VGLUT1 in zebrafish larvae aged 3 and 9 dpf in oxygen-deprived conditions. Results: CA significantly influenced the expression of BDNF and VGLUT1 under OD (p < 0.001). An increase in BDNF expression (p < 0.001) and a decrease in VGLUT1 (p < 0.01) were observed in zebrafish larvae experiencing OD and treated with CA. There was no significant difference in BDNF and VGLUT1 expression across age variations in zebrafish larvae at 3 dpf and 9 dpf in the treatment groups (p > 0.05). CA concentration of 2.5 µg/ml effectively enhanced BDNF and reduced VGLUT1 in 3-9 dpf zebrafish larvae. Conclusion: CA demonstrates potential as a neuroprotective agent, modulating increased BDNF expression and reduced VGLUT1 under OD conditions. These findings lay a foundation for further research in developing therapies for oxygen deficiency.

Effect of Centella asiatica ethanol extract on zebrafish larvae ( Danio rerio) insomnia model through inhibition of Orexin, ERK, Akt and p38.

Background: Insomnia is difficulty initiating or maintaining sleep for at least three nights a week or more and lasting for at least 3 months. One of the molecules that play a role in the circadian rhythm of arousal system is hypocretin/orexin. Orexin activates the p38-MAPK signaling pathway and increases phosphorylated ERK1/2 levels. Centella asiatica (CA) has a role in the signal work of the MAPK/ERK, Akt, and p38 path in many various diseases. Methods: The research method used is true laboratory experimental. The research approach used was randomized control group post-test only. Zebrafish embryos aged 0-7 dpf were used in this study. The treatment group consisted of 5 groups: normal, insomnia, insomnia + 2.5 µg/mL CA, insomnia + 5 µg/mL CA, and insomnia + 10 µg/mL CA. The locomotor motion of zebrafish larvae was observed using Basler cameras on days five-, six- and seven-day post fertilization (dpf), then analyzed by using Western Blot method. Results: The results proved that exposure to CA extract was able to reduce the expression of orexin (91963 ± 9129) and p38 (117425 ± 6398) as an arousal trigger in the sleep-wake cycle, with the most optimal concentration of CA 5 µg/mL. Exposure to CA extract was also able to reduce the expression of ERK (94795 ± 30830) and Akt (60113.5 ± 27833.5) with an optimum concentration of CA 2.5 µg/mL. Conclusion: Exposure to CA extract was able to improve the sleep activity of zebrafish larvae insomnia model by extending the total inactivity time ( cumulative duration) and shortening the duration of first sleep ( latency to first) in light and dark phases through inhibition of orexin, ERK, p38, and Akt.

Modification of the height of a weight drop traumatic brain injury model that causes the formation of glial scar and cognitive impairment in rats.

OBJECTIVE: Traumatic brain injury (TBI) is a chronic, progressive condition associated with permanent disabilities, particularly cognitive impairments. Glial scar formation following TBI is considered a contributing factor to these persistent disabilities. Currently, limited research exists on pharmacological interventions targeting glial scar prevention that require a standard weight drop TBI model for glial scar formation. Since there is no established standard TBI model for glial scar formation, this study aims to validate and modify the height of the weight drop model to identify glial scar formation and cognitive impairments. METHODS: Fifteen male Sprague Dawley rats were randomly divided into sham, WD1, and WD2 groups. The weight drop model with a 10 g load was applied to the right exposed brain of the rats from a height of 5 cm (WD1) and 10 cm (WD2) using a modified Feeney's weight drop device. Cognitive impairments were confirmed using the novel object recognition (NOR) test with ethovision software on day 15. Subsequently, the rats were decapitated on day 16, and GFAP immunohistochemical staining was performed to confirm the presence of glial scarring. RESULTS: The WD1 and WD2 groups exhibited a significant increase in glial scar formation compared to the sham group, with the WD2 group resulting in even more pronounced glial scar formation. Only the WD2 model caused statistically significant cognitive damage. The negative correlation coefficient indicates that an increase in GFAP + cells will decrease the cognitive function. CONCLUSION: Modification of the height of the weight drop model, by dropping a weight of 10 g from a height of 10 cm (WD2 group) onto the right brain exposed of the rat has been proven to induce the formation of a glial scar and cognitive impairment.

Asiatic acid increased locomotor and head width by inducing brain-derived neurotrophic factor in intrauterine hypoxia-exposed zebrafish.

Background: Hypoxia ischemia leads to abnormal behavior and growth. Prenatal hypoxia also decreases brain adaptive potential, which can cause fatal effects such as cell death. Asiatic acid (AA) in Centella asiatica is a neuroprotector through antioxidant and anti-inflammatory activities. Aim: This study aimed to analyze the effect of AA as a neuroprotector against hypoxia during intrauterine development on locomotor activity, head width, and brain-derived neurotrophic factor (BDNF) expression. Methods: The true experimental laboratory research used a posttest control-only design. Zebrafish embryos (Danio rerio) aged 0-2 dpf (days postfertilization) were exposed to hypoxia with oxygen levels reaching 1.5 mg/l. Then, AA was administered at successive concentrations, namely, 0.36, 0.72, and 1.45 µg/ml, at 2 hpf (hours postfertilization), 3, 6, and 9 dpf. Head width, velocity activity, and BDNF expression were observed. Results: Intrauterine hypoxia significantly decreased head width, velocity rate, and BDNF expression (<0.001). Administration of AA at all concentrations and age 9 dpf to zebrafish larvae with intrauterine hypoxia exposure increased head width ( p < 0.0001), velocity (p < 0.05), and relative mRNA expression of BDNF (p < 0.05). Conclusion: AA is potentially neuroprotective to the brain in zebrafish larvae exposed to hypoxia during intrauterine development.

Asiatic acid in Centella asiatica extract towards morphological development in an intermittent hypoxia intrauterine embryo model and molecular prediction pathway of insulin-like growth factor-1 receptor signalling.

Background: Hypoxia during pregnancy generates oxidative stress that alters the growth and development of the human fetus. Insulin-like growth factor-1 (IGF-1) receptors are essential for normal fetal growth. Asiatic acid in Centella asiatica (CA) has antioxidant properties to prevent growth impairment in hypoxia. Aims: This study aimed to investigate the effect of asiatic acid on the morphological development of an intermittent hypoxia (IH) zebrafish embryo model and analyze molecular docking prediction in IGF-1 receptor (IGF-1R) signaling. Methods: Embryos of zebrafish at 2 hours postfertilization (hpf) were assigned to control negative (C), IH, and combination IH and CA extract groups consisting of 1.25 (IHCA1), 2.5 (IHCA2), and 5 (IHCA3) µg/ml. Hypoxia treatment (conducted 4 hours/day) and CA extract were administered for 3 days (2-72 hpf). The parameters of body length and head length were evaluated at 3, 6, and 9 days postfertilization (dpf). The data were analyzed by a two-way analysis of variance (p < 0.05). Molecular docking was performed to explore the binding affinity of asiatic acid to IGF-1R by Molegro Virtual Docker ver.5 software. Results: The body length and head length of embryos in the IH and treatment groups (IHCA) were shorter than those in the control group at 3 dpf (p < 0.05). However, the body length was more prolonged in the IHCA1 group, but the head length was longer in the IHCA2 group than in the IH group at 6 and 9 dpf. Molecular docking showed the reliable interaction of asiatic acid with IGF-1R signaling in an IH animal model. Conclusion: The administration of CA extract benefits IH through the development and growth of zebrafish embryos at a dose of 2.5-5 µg/ml. Asiatic acid has a binding affinity for IGF-1R signaling.

Aeromonas hydrophila induction method in adult zebrafish (Danio rerio) as animal infection models.

Background and Aim: Zebrafish are frequently used as model organisms in scientific research as their genes mirror those of humans. Aeromonas hydrophila bacteria can infect humans and animals, mainly fish. This study aimed to identify the concentration and route of A. hydrophila infection in adult zebrafish. Zebrafish had been used as a challenge test by analyzing their hematological profiles, blood glucose levels, and survival rates. Materials and Methods: Induction of cell supernatant free (CSF) from A. hydrophila bacteria in adult zebrafish was carried out via bath immersion (BI), intraperitoneal injection (IPI), intramuscular injection (IMI), and healthy zebrafish as a control (C). The bacterial concentrations were 107, 109, and 1011 colony-forming units (CFU)/mL. At 24 h post-infection, the outcomes of infection were evaluated based on survival rates, hematological profiles, and blood glucose levels. A one-way analysis of variance with a confidence level of 95% was employed to examine the data. Results: In the BI, IPI, and IMI treatment groups, the survival rate of the fish reached a peak of 100%, 22%-100%, and 16%-63%, respectively, compared with the injection technique. In the IMI2 group, a 109 CFU/mL bacterial concentration was determined to correspond to the lethal dosage 50. All infection groups had lower erythrocyte and hemoglobin counts but higher leukocyte counts than the control group. The blood sugar levels of the healthy and infected groups were not significantly different. Conclusion: The route of A. hydrophila infection through Intramuscular injection with a concentration of 109 CFU/mL indicated a high performance compared to other techniques. This method could be developed as a reproducible challenge test.

Metabolomics Study Reveals Biomarker L-Proline as Potential Stress-Protectant Compound for High-Temperature Bioethanol Fermentation by Yeast Pichia kudriavzevii 1P4.

High-temperature ethanol fermentation (> 40 °C) can be applied as effective bioprocess technology to increase ethanol production. Thermotolerant yeast Pichia kudriavzevii 1P4 showed the ability to produce ethanol at optimum 37 °C. Thus, this study evaluated the ethanol productivity of isolate 1P4 at high-temperature ethanol fermentation (42 and 45 °C) and the identification of metabolite biomarkers using untargeted metabolomics with liquid chromatography-tandem mass spectrometry (LC-MS/MS). 1P4 showed tolerance to temperature stress up to 45 °C and thus relevant for high-temperature fermentation. As measured by gas chromatography (GC), bioethanol production of 1P4 at 30, 37, 42, and 45 °C was 5.8 g/l, 7.1 g/l, 5.1 g/l, and 2.8 g/l, respectively. The classification of biomarker compounds was based on orthogonal projection analysis to latent structure discriminant analysis (OPLS-DA), resulting in L-proline being a suspected biomarker compound for isolate 1P4 tolerance against high-temperature stress. Indeed, supplementation of L-proline on fermentation medium supported the growth of 1P4 at high temperatures (> 40 °C) than without L-proline. The bioethanol production with the addition of the L-proline resulted in the highest ethanol concentration (7.15 g/l) at 42 °C. Supplementation of L-proline as a stress-protective compound increased ethanol productivity at high-temperature fermentation of 42 and 45 °C by 36.35% and 83.33%, respectively, compared without the addition of L-proline. Preliminary interpretation of these results indicates that bioprocess engineering through supplementation of stress-protective compounds L-proline increases the fermentation efficiency of isolate 1P4 at higher temperatures (42 °C and 45 °C).

Methanol extract of Black soldier fly (Hermetia illucens) prepupae against Aeromonas and Staphylococcus aureus bacteria in vitro and in silico.

Background: Staphylococcus and Aeromonas bacteria are pathogens in humans and animals. The therapy disrupts the virulence structure of the bacteria, resulting in bacterial death. Currently, chemical drugs have resulted in many resistant bacteria, so it is necessary to find alternative natural materials that are not toxic and do not quickly induce resistance. Aims: This study aimed to analyze the potential of methanol extract from Black soldier fly (BSF) prepupae as an antibacterial agent against Staphylococcus aureus and Aeromonas through in silico and in vitro tests. Methods: The BSF prepupae methanol extract was analyzed for protein and fatty acid contents. Disc diffusion method, minimal inhibitory concentration, and minimum bactericidal concentration test were used for in vitro tests against Staphylococcis and Aeromonas. Molecular docking of the active ingredients (defensin, chitin, and chitosan as well as fatty acids) in BSF was downloaded from the NCBI database and docked by the Hex Cuda version 8.0 program with Correlation type parameters Shape + Electro and Grid Dimension version 0.6. Docking results were analyzed using the Discovery Studio program version 21.1.1. Results: The highest fatty acid contents in the extract were palmitic acid and myristic acid. Methanol extract from BSF prepupae acted as a bactericidal agent against S. aureus at a concentration of 320 mg/ml, in contrast to Aeromonas, which still showed bacterial growth. The results of the in silico test showed that defensin-aerolysin and defensin-hemolysin was bound to the same active site area. However, the amount of binding energy produced by 69-Defensin-83-aerolysin was higher than all defensin types in BSF against Aeromonas. Chitin and chitosan showed a bond on the active site of aerolysin and hemolysin, but chitosan had a stronger bond than chitin. In silico study also showed the strongest binding affinity of BSF fatty acids to isoleucyl-tRNA synthetase of S. aureus. Conclusion: The study showed that methanol extract from BSF prepupae had potential capability as an antibacterial agent against S. aureus than Aeromonas in vitro and in silico.

In Vitro Insulin Resistance Model: A Recent Update.

Insulin resistance, which affects insulin-sensitive tissues, including adipose tissues, skeletal muscle, and the liver, is the central pathophysiological mechanism underlying type 2 diabetes progression. Decreased glucose uptake in insulin-sensitive tissues disrupts insulin signaling pathways, particularly the PI3K/Akt pathway. An in vitro model is appropriate for studying the cellular and molecular mechanisms underlying insulin resistance because it is easy to maintain and the results can be easily reproduced. The application of cell-based models for exploring the pathogenesis of diabetes and insulin resistance as well as for developing drugs for these conditions is well known. However, a comprehensive review of in vitro insulin resistance models is lacking. Therefore, this review was conducted to provide a comprehensive overview and summary of the latest in vitro insulin resistance models, particularly 3T3-L1 (preadipocyte), C2C12 (skeletal muscle), and HepG2 (liver) cell lines induced with palmitic acid, high glucose, or chronic exposure to insulin.

The Effect of Salmonella AvrA Infection on Colon Cancer: An in-silico Study.

OBJECTIVE: Colorectal cancer (CRC) is one of the main causes of morbidity and mortality due to cancer. The purpose of this in-silico study was to examine the relationship of chronic infection mechanisms caused by Salmonella Anti virulence agent A (AvrA) to gene mutations in the carcinogenic process of CRC. METHODS: Gene expression data on the mouse colon was obtained from the GSE22215 dataset | Gene Expression Omnibus (GEO). Adjusted p-value was calculated using Benjamini & Hochberg False Discovery Rate (FDR<0.01). Gene expression in colon adenocarcicoma tumors was obtained from The Cancer Genome Atlas's (TCGA) Genomic Data Commons (GDC) dataset containing 458 colon tumor samples. RESULT: Expressions of MLH1, MSH2, EPCAM, APC, and PMS2 in cases of colon adenocarcinoma tumor showed a correlation with genes that underwent changes due to Salmonella AvrA infection. Among the gens of interest, EPCAM was the gene that had the highest correlation compared to other genes (MLH1, MSH2, APC, and PMS2) (n= 514, Gene r-p value < 0.01 =22355). There were 514 genes that had a correlation with cases of AvrA infection. Tumor Necrosis Factor (TNF), which is a gene that is upregulated in AvrA infection and correlates negatively with EPCAM, had the highest BC value compared to other gens (p= 0.0000768). Survival probability showed that EPCAM was highly expressed and it can increase survival time. In addition to TNF, our study indicated that IL1B (p= 0.000419), S100A8 (p= 2.02E-05), S100A9 (p=0.000419) correlated with the gene of interest. CONCLUSION: Late Salmonella AvrA infection affects the expression of genes involved in inflammation in colorectal cancer samples.

Bipartite graph search optimization for type II diabetes mellitus Jamu formulation using branch and bound algorithm.

Jamu is an Indonesian traditional herbal medicine that has been practiced for generations. Jamu is made from various medicinal plants. Each plant has several compounds directly related to the target protein that are directly associated with a disease. A pharmacological graph can form relationships between plants, compounds, and target proteins. Research related to the prediction of Jamu formulas for some diseases has been carried out, but there are problems in finding combinations or compositions of Jamu formulas because of the increase in search space size. Some studies adopted the drug-target interaction (DTI) implemented using machine learning or deep learning to predict the DTI for discovering the Jamu formula. However, this approach raises important issues, such as imbalanced and high-dimensional dataset, overfitting, and the need for more procedures to trace compounds to their plants. This study proposes an alternative approach by implementing bipartite graph search optimization using the branch and bound algorithm to discover the combination or composition of Jamu formulas by optimizing the search on a plant-protein bipartite graph. The branch and bound technique is implemented using the search strategy of breadth first search (BrFS), Depth First Search, and Best First Search. To show the performance of the proposed method, we compared our method with a complete search algorithm, searching all nodes in the tree without pruning. In this study, we specialize in applying the proposed method to search for the Jamu formula for type II diabetes mellitus (T2DM). The result shows that the bipartite graph search with the branch and bound algorithm reduces computation time up to 40 times faster than the complete search strategy to search for a composition of plants. The binary branching strategy is the best choice, whereas the BrFS strategy is the best option in this research. In addition, the the proposed method can suggest the composition of one to four plants for the T2DM Jamu formula. For a combination of four plants, we obtain Angelica Sinensis, Citrus aurantium, Glycyrrhiza uralensis, and Mangifera indica. This approach is expected to be an alternative way to discover the Jamu formula more accurately.

Decreasingα-synuclein aggregation by methanolic extract of Centella asiatica in zebraifsh Parkinson’s model

Objective:To observe the effects of Centella asiatica (C. asiatica) methanolic extract onα-synuclein aggregation and its expression in rotenone-exposed zebrafish. Methods: Zebrafish (Danio rerio) were exposed to 5 μg/L rotenone for 28 days and co-incubated with 2.5, 5.0 and 10.0 μg/mL of C. asiatica methanolic extract. The medium was changed every 48 h for maintain the concentration of rotenone and extract. After 28 days zebrafish were sacrificed on the ice block and protein was isolated from zebrafish brain for ELISA of dopamine and Western blotting of α-synuclein. Immunohistochemistry was conducted to observe the α-synuclein expressions from histopathological preparation of zebrafish brain. The head were soaked in 10%formaline for less than 24 h and embedded onto paraffin block, then sliced for immunohistochemistry using antiα-synuclein antibody. We also measured zebrafish motility for 5 min in each week. Results:C. asiatica has important bioactive compounds such as asiaticoside that has anti-inflammatory and antioxidant properties. It may inhibit cascade reaction due to oxidative stress induced by rotenone. Decreasing reactive oxygen species proposed probability of radical attack toα-synuclein protein that caused aggregation and increase of its expression. The motility of zebrafish was also maintained in C. asiatica groups due to the increasing dopamine level in rotenone-induced zebrafish. High level of reactive oxygen species inactivated enzyme for dopamine synthesis such as tyrosine hydroxylase, and oxidized dopamine itself. Oxidized dopamine increasedα-synuclein aggregation. Thus, the dopamine level decreased in rotenone-induced zebrafish, but C. asiatica increased dopamine level. Conclusions: C. asiatica has a potential to be developed as an anti-Parkinson's disease treatment due to its capability for minimized the sign of Parkinson’s such asα-synuclein aggregation and expression, increasing motility and dopamine as well.