Profibrotic Inflammatory Cytokines and Growth Factors Are Predicted as the Key Targets of Uncaria gambir (Hunter) Roxb. in Keloids: An Epistatic and Molecular Simulation Approach.

Keloid is characterized as the fibrotic tissue resulting from the increase of fibroblast activity. Uncaria gambir (Hunter) Roxb. possesses bioactive compounds that have potential as antifibrotic agents, while the mechanism of action in keloid has not yet been elucidated. The aim of this study was to investigate the interaction of gambir bioactive compounds with keloid target proteins using an epistatic and molecular simulation approach. The known bioactive compounds of gambir targets and keloid-related protein targets were screened using databases. The network was constructed and analyzed to obtain the core protein targets. The targets were enriched to describe the Gene Ontology (GO) and pathway related to the proteins. Eleven targets were defined as the main targets of gambir bioactive compounds related to keloid disease. Gambiriin C, Isogambirine, and Procyanidin B1 were identified as the most promising compounds with the highest binding energy to transforming growth factor beta 1 (TGFß1), AKT serine/threonine kinase 1 (AKT1), and matrix metallopeptidase 1 (MMP1) as the target proteins. GO enrichment and pathway analysis found that gambir bioactive compounds may act on keloid-related target proteins to regulate cell proliferation, migration, transcription, and signal transduction activity via profibrotic cytokine and growth factor signaling pathways. This study provides a reference for potential targets, compounds, and pathways to explain the mechanism of gambir against keloid.

HIF2-α Expression in CML Patients Receiving Hydroxyurea Prior to Imatinib That Achieved Major Molecular Response (MMR) versus in Those Not Achieving MMR.

Introduction: Currently, Imatinib (IM) which is a Tyrosine Kinase Inhibitor (TKI), is the main treatment for patients with chronic myeloid leukemia (CML). Major molecular response (MMR) is used as therapeutic response. Resistance to IM may be caused by hypoxia which is regulated by hypoxia inducible factor (HIF) 2-α. The role of HIF2-α is currently not researched extensively. This study aimed to analyse the differences in HIF-2α expression between chronic phase CML patients that achieved MMR and those that did not achieve MMR. Methods: This study used a cross-sectional method which analysed secondary data from whole blood samples in chronic phase CML patients aged 18-60 years that received hydroxyurea (HU) before IM, aged 18-60 years, received IM therapy for more than 12 months, and were willing to participate in the study. The exclusion criteria for this study were patients who were receiving IM at a dose of more than 400 mg/day. HIF-2α protein expression was examined using the enzyme-linked immunosorbent assay (ELISA) method. Differences between HIF-2α protein expression in groups that achieved MMR versus not achieving MMR was analysed using the Mann-Whitney test. Results: A total of 79 subjects were obtained. The median HIF-2α was 90.56 pg/mg protein (3.01-4628.74). There was no statistically significant difference in expression of HIF-2α in the group that reached MMR and did not reach MMR, namely 123.45 pg/mg protein and 89.25 pg/mg protein respectively (p 0.718). Conclusion: This study found no statistically significant difference between HIF-2α expression level and MMR achievement of chronic phase CML patients who received HU before IM therapy.

Inhibition of ALA dehydratase activity in heme biosynthesis reduces cytoglobin expression which is related to the proliferation and viability of keloid fibroblasts.

The aim of this study was to analyze the effect of heme synthesis inhibition on cytoglobin expression and its correlation with keloid fibroblast viability and proliferation. The study was conducted on primary culture of keloid fibroblasts. Heme synthesis in keloid fibroblasts was inhibited using succinyl acetone. We measured amino levulinic acid dehydratase (ALAD) enzyme activity using a colorimetric method; cytoglobin mRNA expression using qRT-PCR, cytoglobin protein expression using ELISA and immunocytochemistry, fibroblast viability using the MTT test; and fibroblast proliferation using BrdU test. The results showed that the ALAD enzyme activity level was lower in the keloid fibroblasts treated with succinyl-acetone (SA, 1, 2.5, and 5 mM) than in the control. The cytoglobin mRNA and protein expressions level were significantly lower in the keloid fibroblasts cultured with 2.5 mM and 5 mM SA than in the control and 1 mM SA. The viability and proliferation of the keloid fibroblasts decreased when the SA concentration was increased. In conclusion, the use of succinyl acetone at a concentration of 1; 2.5; and 5 mM caused decrease ALAD enzyme activity which indicated the inhibition of the heme synthesis. Inhibition of heme synthesis can affect cytoglobin expression, which correlates with the viability and proliferation of keloid fibroblasts.

Clinical evaluation of intralesional umbilical cord-derived mesenchymal stem cells, conditioned medium and triamcinolone acetonide injection for keloid treatment: A pilot study.

Topical keloid therapy is performed with triamcinolone acetonide (TA) intralesional injection. However, the recurrence rate is high with various side effects. Mesenchymal stem cells (MSCs) have high proliferative abilities and reduce the activity and proliferation of fibroblast cells in keloids. To overcome the costs and limitations, conditioned medium (CM) is used. This study aims to evaluate feasibility of intralesional injection of umbilical cord MSC (UC-MSC) and conditioned medium (UC-CM) compared to TA for keloid therapy. Twenty-four patients with keloids who met the inclusion criteria were included, randomized into three treatment groups and then got assessed for the sociodemographic data, keloid volume, histopathology (type 1:3 collagen ratio), interleukin-10 (IL-10) levels and Patient and Observer Scar Assessment Scale (POSAS) score during visits. Largest volume regression occurred in the UC-MSC group, followed by UC-CM and then the TA group (UC-MSC: 45.32% ± 2.61%; UC-CM: 43.61% ± 3.67%; TA: 28.34% ± 3.81%; p = 0.003). Similar pattern was also observed in increase in IL-10 levels, the decrease in POSAS scores and the reduction of type 1:3 collagen ratio. Hence, UC-MSC and UC-CM are promisingly more effective than TA for keloid therapy, showcasing their superiority in reducing keloid volume, symptoms and type 1:3 collagen ratio, as well as increasing the levels of IL-10.

Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb).

Background: Cytoglobin (Cygb) is a relatively newly identified globin protein that acts as an oxygen transporter in tissues like hemoglobin (Hb) in erythrocytes and myoglobin (Mb) in muscles. The natural oxidation of the Fe2+ ion in its heme group into metglobin (globin-Fe3+) made the loses of oxygen binding functions. It is known metHb and metMb can be reduced enzymatically using diaphorase or cyb5r3. However, metCygb reductase had not been previously identified. This study aims to analyze the reducing activity of bovine diaphorase on metCygb. Methods: Diaphorase was isolated from bovine erythrocyte and purified using gel filtration and cationic-exchanger chromatography. Its purity was verified by SDS-PAGE and western blot (WB). The metCygb was obtained from Cygb oxidation with potassium ferrocyanide and its reducing activity was determined by spectroscopy. Results: The diaphorase (MW=30.09 kDa) was purified 10.77-fold from crude enzyme with specific activity against metHb 8.479 U/mg. The purity was confirmed by WB using primary antibody anti-cyb5r3. The purified enzyme reduced metCygb at 0.785 µgmin-1, which was 13.7 times less than the Vmax of metHb. Discussion: In conclusion, the purified diaphorase from bovine erythrocytes did not significantly reduce metCygb rather than metHb, a natural substrate in cells.

Fibropreventive and Antifibrotic Effects of Uncaria gambir on Rats with Pulmonary Fibrosis.

Pulmonary fibrosis causes scar tissue formation that disrupts the functioning of the lungs. Uncaria gambir (Hunter) Roxb (hereafter gambir)-a plant native to West Sumatra in Indonesia-contains flavonoid (+)-catechin, which has strong antioxidant activity and can be used to combat pulmonary fibrosis. This random in vivo experimental study analyzed the antifibrotic effect of gambir on the lungs of rats with bleomycin-induced fibrosis. The subjects were 10 groups of 10-week-old male rats weighing around 200-250 g. All groups were terminated at the end of the seventh week or on day 50. The lungs were cleaned, and tissues were taken to analyze inflammatory cell counts and TGF-ß1 levels using bronchoalveolar lavage (BAL) with ELISA; type I collagen and tissue inhibitor of metalloproteinase 1 (TIMP-1) levels using immunohistochemistry (IHC); and activation of NF-κB using ELISA and Western blot assays. The most severe histopathological characteristic based on the modified Ashcroft score was in the bleomycin group (BG), whereas the mildest was in the 262 mg/kg of the bodyweight antifibrotic gambir-dosed group (AF G262). The results showed a significant difference in the BAL inflammatory cell count (p=0.017; p < 0.05). AF G262 differed most from the other antifibrotic groups in terms of the number of inflammatory cells (0.63), TGF-ß1 levels (3.80), and NF-κB levels (0.48), followed by the 131 mg/kg of the bodyweight antifibrotic gambir-dosed group (AF G131), which also differed most from other antifibrotic groups in terms of NF-κB (0.48), TIMP-1 (11.74), and collagen I (14.50) levels. Western blot analysis showed that the fibropreventive and antifibrotic groups had a specific band size of p65, whereas no specific band binding existed in the control group. This study concluded that the administration of AF G262 could improve fibrosis by lysing the extracellular matrix (ECM) in rat lungs.

Resuscitation of hemorrhagic shock using normal saline does not damage the glycocalyx in the immediate resuscitation phase.

OBJECTIVES: The objectives were to study the effect of aggressive resuscitation using normal saline on hemodynamics, serum atrial natriuretic peptide (ANP), syndecan-1 (marker of endothelial glycocalyx shedding), and extravascular lung water index (ELWI) following hemorrhagic shock. METHODS: Eleven male piglets (Sus scrofa) underwent blood drawing to create 20% drop in mean arterial pressure (MAP). Two-phase resuscitation was performed: Phase 1 using normal saline of an equal volume of blood drawn to create shock and Phase 2 using 40 ml/kg BW of normal saline to simulate hypervolemia and hemodilution. Heart rate, MAP, cardiac index (CI), systemic vascular resistance index, oxygen delivery (DO2), global end-diastolic volume index, ELWI, hemoglobin (Hb), lactate, ANP, and syndecan-1 at each phase and up to 60 min following Phase 2 resuscitation were recorded. RESULTS: Phase 2 resuscitation significantly decreased Hb concentration (P = 0.006), however, DO2 was maintained (P = 1.000). CI increased from shock to Phase 1 (P = 0.029) and further increase in Phase 2 resuscitation (P = 0.001). Overall, there was a transient increase of ANP following Phase 1 resuscitation, from 85.20 ± 40.86 ng/L at baseline to 106.42 ± 33.71 ng/L (P = 0.260). Serum syndecan-1 and ELWI change at all phases were not significant. CONCLUSIONS: We demonstrate compensatory protective mechanism despite overzealous fluid resuscitation. Compensatory increased CI despite decreased Hb maintained DO2. In the absence of inflammation, serum ANP did not increase significantly, no glycocalyx shedding occurred, subsequently no change in ELWI. We show that factors other than volume overload are more dominant in causing glycocalyx shedding.

Melatonin effect on hypoxia inducible factor-1α and clinical response in patients with oral squamous cell carcinoma receiving neoadjuvant chemotherapy: A randomized controlled trial.

CONTEXT: Chemoresistance is a major issue in patients with locally advanced oral squamous cell carcinoma (OSCC). In this study, we evaluated the effectiveness of melatonin in conjunction with neoadjuvant chemotherapy (NC) on hypoxia-inducible factor-1α (HIF-1α) expression and clinical response in locally advanced OSCC patients. AIMS: To study the effects of melatonin on HIF-1α expression and its effect on the clinical response of patients with locally advanced OSCC. SETTINGS AND DESIGN: A randomized controlled trial was conducted, wherein patients were recruited from several hospitals in Jakarta, Indonesia. Patients were randomized into two groups using computerized block randomization. SUBJECTS AND METHODS: Both groups were given NC, with treatment group receiving melatonin. Outcomes measured in this study were HIF-1α expression from tissue samples and clinical response based on the RECIST 1.1 criteria. Twenty-five patients completed the study protocol and were included in the data analysis. STATISTICAL ANALYSIS USED: Shapiro-Wilk test was used to test the data normality. For data with normal distribution, we conducted an independent t-test to compare between the two groups. Data with abnormal distribution were analyzed using Mann-Whitney U-test. The mean difference between the two groups was analyzed using Shapiro-Wilk normality test. RESULTS: Our study showed a significant decrease in HIF-1α expression in the melatonin group compared to the placebo group (P < 0.05, relative risk 3.08). However, the degree of reduction of HIF-1α expression in the melatonin group did not differ significantly (P = 0.301). CONCLUSIONS: Our study showed that melatonin administered at 20 mg/day could reduce the expression of HIF-1α and residual tumor percentage, but did not affect the clinical response in OSCC patients.

Is the Mitochondrial Function of Keloid Fibroblasts Affected by Cytoglobin?

BACKGROUND: A keloid is a benign skin tumour characterised by excessive proliferation of fibroblasts, a process that requires a sufficient amount of energy. The energy needs are associated with adequate oxygen (O2) flow and well-functioning mitochondria. It is known that cytoglobin (CYGB) has a function in O2 distribution. The aim of the present study was to explore whether the inhibition of CYGB expression caused impaired mitochondrial function of keloid fibroblasts. METHODS: An in vitro study was conducted on a keloid fibroblast derived from our previous study. The study was carried out in the laboratory of the Biochemistry & Molecular Biology Department, Faculty of Medicine, Universitas Indonesia (FMUI), from July to December 2018. CYGB expression was inhibited by small interfering ribonucleic acid (siRNA) and CYGB. Analysis of mitochondrial function was observed through peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), a mitochondrial biogenesis marker and the activity of the succinate dehydrogenase (SDH) enzyme in mitochondria. RESULTS: The CYGB gene and protein were downregulated after treatment with CYGB siRNA. Inhibition of CYGB expression with siRNA also tended to decrease the levels of PGC-1α messenger ribonucleic acid (mRNA) and protein, as well as SDH enzyme activity. CONCLUSION: Inhibition of CYGB expression with siRNA tended to decrease mitochondrial biogenesis and function. This may be useful for understanding the excessive proliferation of fibroblasts in keloids and for development of treatment for keloids.

Phagocytosis and the antigen-processing abilities of macrophages derived from monocytes in spinal tuberculosis patients.

This study examined the hypothesis that there is an impairment of macrophageal function in spinal TB. We examined macrophageal functions in spinal TB patients. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of five spinal TB patients and five healthy persons as control. The isolated monocytes were cultured with stimulation of macrophage colony-stimulating factor (M-CSF) for seven days for maturation. The phagocytic ability of the macrophages derived from monocytes was measured. Also, nitric oxide (NO), myeloperoxidase (MPO), beta-glucuronide, and acid phosphatase activity was investigated. We found that the monocytes collected from patient PBMCs were significantly fewer than those of the control group (2992.103 vs. 6474.103 (cells/mL)). There were also fewer macrophages that had adhered to sheep red blood cells (SRBC) (598.103 vs. 264.103 (cells/mL)). However, NO production (2346 vs. 325.17 (µmol/gram of protein)), and the MPO (570.7 vs. 17.4 (unit/mg), beta-glucuronide (0.149 vs. 0.123 (µmol/hour/100 mg of protein)), and acid phosphatase activities (1776.9 vs. 287.9 (µmol/hour/100 mg of protein)) of the macrophages in the spinal TB group were markedly higher than in the healthy group. Despite the low adhesion to foreign bodies, the intracellular processing of TB macrophages, including oxidative activity and lysosome function, was significantly high. These results suggested the impairment of macrophageal function in spinal TB. Possibly, there is a dominance of innate non-specific immunity in spinal TB infection.

The Differential Effects of Propylthiouracil and Methimazole as Graves' Disease Treatment on Vascular Atherosclerosis Markers: A Randomized Clinical Trial.

Background: Hyperthyroidism is related to vascular atherosclerosis. Propylthiouracil (PTU) and methimazole, other than their antithyroid effects, may have different mechanisms in preventing atherogenesis in Graves' disease. Objective: This study aimed to investigate the effect of antithyroid drugs on markers of vascular atherosclerosis in Graves' hyperthyroidism. Methods: This study was a single-blind, randomized clinical trial conducted on 36 patients with Graves' disease in Cipto Mangunkusumo General Hospital, Jakarta, Indonesia, from June 2019 until July 2020. Graves' disease was diagnosed from clinical manifestation of hyperthyroidism with diffuse goiter and then confirmed by thyroid stimulation hormone (TSH), free T4 (fT4), and TSH-receptor antibody (TRAb) measurements. Participants were randomly assigned to either a PTU or a methimazole treatment group and followed up for 3 months. Markers of vascular atherosclerosis were represented by adhesion molecules [intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin], carotid artery stiffness [pulse wave velocity (PWV)], and thickness [carotid intima media thickness (cIMT)]. Results: By the end of the study, 24 participants reached euthyroid condition (13 from the PTU group and 11 from the methimazole group). After 3 months of follow-up, in the PTU group, we noticed an improvement of ICAM-1 [pretreatment: 204.1 (61.3) vs. posttreatment: 141.6 (58.4) ng/ml; p = 0.001], VCAM-1 [837 (707-977) vs. 510 (402-630) ng/ml; p < 0.001] and E-selectin [32.1 (24.1-42.7) vs. 28.2 (21.6-36.8) ng/ml; p = 0.045] in the PTU group. In the methimazole group, only VCAM-1 improvement [725 (565-904) vs. 472 (367-590); p = 0.001] was observed. Meanwhile, we found no significant changes in PWV or cIMT in either group. Conclusion: Antithyroid treatment in Graves' disease leads to improvement in adhesion molecules, with a lesser effect on methimazole, whereas there were no significant changes in PWV or cIMT. PTU may have a better mechanism compared with methimazole in terms of improving adhesion molecules.

Oral and Topical Centella asiatica in Type 2 Diabetes Mellitus Patients with Dry Skin: A Three-Arm Prospective Randomized Double-Blind Controlled Trial.

INTRODUCTION: Uncontrolled diabetes mellitus (DM) is related to skin disorders, particularly dry skin. Pathogenesis of dry skin in type 2 diabetes mellitus (T2DM) rises from the chronic hyperglycemia causing an increase in advanced glycation end-products (AGEs), proinflammatory cytokines, and oxidative stress. Combination of oral and topical Centella asiatica (CA) is expected to treat dry skin in T2DM patients more effectively through decreasing N(6)-carboxymethyl-lysine (CML) and interleukin-1α (IL-1α) and increasing superoxide dismutase (SOD) activity. METHODS: A three-arm prospective, double-blind, randomized, controlled study was performed to evaluate the efficacy of the oral and topical CA extract in 159 T2DM patients with dry skin. The subjects were divided into the CA oral (CAo) 2 × 1.100 mg + CA topical (CAt) 1% ointment group, oral placebo (Plo) + CAt group, and Plo and topical placebo (Plt) group. Dry skin assessment was performed on day 1, 15, and 29, while evaluation of CML, IL-1α, and SOD activity was on day 1 and 29. RESULT: Effectivity of CAo + CAt combination was assessed based on HbA1c and random blood glucose (RBG). In well-controlled blood glucose, on day 29, the percentage of SRRC decrement was greater in the CAo + CAt group compared to the control group (p = 0.04). SCap value in the CAo + CAt group was greater than that in the control group (p = 0.01). In the partially controlled blood glucose, increment of SOD activity in the CAo + CAt group was greater than that in the control group (p = 0.01). There were medium-to-strong correlation between CML with SOD (r = 0.58, p < 0.05) and IL-1α with SOD (r = 0.70, p < 0.05) in well-controlled blood glucose. Systemic and topical adverse events were not significantly different between groups. CONCLUSION: CAo and CAt combination can be used to significantly improve dry skin condition through increasing SOD activity in T2DM patients with controlled blood glucose.

Determining a critical threshold for G6PD activity below which red blood cell response to oxidative stress is poor.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme disorder in the world. Its main function is to generate NADPH that is required for anti-oxidative pathway in the cells especially in red blood cells (RBC). G6PD deficiency is X-linked and thus subject to random X-chromosome inactivation in women giving them mosaic expression of G6PD activities in their individual cells. This phenomenon makes it difficult for diagnosis with the currently available G6PD qualitative diagnostic tests. With the rolling out of newly marketed anti-malarial drug tafenoquine, which has a long half-life, screening for G6PD deficiency becomes a necessity where those with < 70% G6PD activity cannot receive this drug. Thus, evidence for a quantitative cut-off for G6PD activity is needed to ensure safe drug administration. METHODS: RBC models were developed to analyse the effect of oxidant on RBC oxidative markers namely total glutathione (GSH)and malondialdehyde (MDA). G6PD activity was measured using quantitative assay from Trinity Biotech and was correlated with cytofluorometric assay. RBC from two G6PD heterozygous women with different G6PD activities were also analysed for comparison. RESULTS: There was a negative correlation between G6PD activity and CuCl concentration and a strong association between G6PD activities and proportion of G6PD normal RBC in CuCl-treated models and in ex vivo RBC. However, in terms of oxidative stress markers analyses, unlike the hypothesis where the lower G6PD activity, the higher MDA and the lower GSH level, the CuCl RBC model showed that in low G6PD activities (10-30%) cells, the MDA level is lower compared to the rest of the models (p < 0.05). The ex vivo models however were in line with the hypothesis, although the result was not significant (p = 0.5). There was a significant difference between RBC with < 60% and those with > 80% G6PD activities in CuCl RBC model, but not in ex vivo RBC (p = 0.5). Genotyping heterozygous subjects showed G6PDViangchan variant with 2.97 U/gHb (33% activity) and 6.58 U/gHb (74% activity). CONCLUSIONS: The GSH analysis has pointed to the 60% G6PD activity cut-off and this data is supportive of the old World Health Organization threshold for intermediate upper limit of 60% G6PD activity. However, there are significant limitations in using MDA assay with CuCl RBC model because the RBC was already stressed due to the copper treatment and thus present a different result when compared to the ex vivo model.

Effect of melatonin supplementation in combination with neoadjuvant chemotherapy to miR-210 and CD44 expression and clinical response improvement in locally advanced oral squamous cell carcinoma: a randomized controlled trial.

BACKGROUND: Squamous cell carcinoma of the oral cavity (OSCC) is the sixth most common malignancy. Surgery is mainstay treatment for oral cancers. Surgery in locally advanced OSCC presents many challenges primarily because the head and neck have critical structures that can be damaged by tumor or treatment. It is thought that neoadjuvant chemotherapy (NC) in locally advanced OSCC is able to shrink tumor size. Chemoresistancy is a problem due to hypoxic microenvironment characterized by increased expression of HIF-1α. It is also regulated by miR-210 as well as increased expression of CD44 and CD133. Melatonin has a powerful antioxidant and oncostatic effects that are expected to improve tumor hypoxia and clinical response. Fifty patients with OSCC were included and randomized. miR-210 and CD44 expression were measured before and after intervention using qRT-PCR absolute quantification, and clinical response was evaluated according to RECIST 1.1 criteria. This study aims to determine the effect of melatonin in improving the clinical response of patients with locally advanced oral squamous cell carcinoma (OSCC) after neoadjuvant chemotherapy to miR-210 and CD44 expression. RESULTS: Melatonin administration reduced miR-210 levels but not significant (p = 0.767). CD44 expression also decreased in the melatonin group compared with placebo yet was not significant (p = 0.103). There was a decrease in the expression of miR-210 and CD44 followed by a decrease in the percentage of residual tumor but not significant (p = 0.114). CONCLUSION: In OSCC, the addition of 20-mg melatonin to neoadjuvant chemotherapy (NC) reduced the expression of miR-210 and CD44 and decreased the percentage of tumor residue; however, no statistically significant result was observed. TRIAL REGISTRATION: This study is registered to ClinicalTrials.gov under trial registration number: NCT04137627 with date of registration on October 22, 2019-retrospectively registered, accessible from: https://clinicaltrials.gov/ct2/show/NCT04137627.

High expressions of the cytoglobin and PGC-1α genes during the tissue regeneration of house gecko (Hemidactylus platyurus) tails.

BACKGROUND: The tissue regeneration process requires high oxygen and energy levels. Cytoglobin (Cygb) is a member of the globin family, which has the ability to bind oxygen, plays a role in dealing with oxidative stress, and carries oxygen into the mitochondria. Energy production for tissue regeneration is associated with mitochondria-especially mitochondrial biogenesis. The peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha protein helps to regulate mitochondrial biogenesis. House geckos (Hemidactylus platyurus) are reptiles that have the ability to regenerate the tissue in their tails. House geckos were selected as the animal models for this study in order to analyze the association of Cygb with oxygen supply and the association of PGC-1α with energy production for tissue regeneration. RESULTS: The growth of house gecko tails showed a slow growth at the wound healing phase, then followed by a fast growth after wound healing phase of the regeneration process. While Cygb mRNA expression reached its peak at the wound healing phase and slowly decreased until the end of the observation. PGC-1α mRNA was expressed and reached its peak earlier than Cygb. CONCLUSIONS: The expressions of both the Cygb and PGC-1α genes were relatively high compared to the control group. We therefore suggest that Cygb and PGC-1α play an important role during the tissue regeneration process.

Hypoxia and autophagic response of obese adult rat adipocytes which differ in nutritional state during childhood.

The prevalence of obesity in adults is increasing worldwide, which is problematic since obesity is associated with degenerative diseases. Nowadays Indonesia is facing an interesting phenomenon since there are adults who have been obese since childhood and others who conversely were undernourished while young. The biological differences of these two types of obesities are not well understood. This study aims to analyse the difference in the size and number of visceral adipocytes, HIF-1α, HIF-2α and MAP1LC3A/LC3 in obese adult rat groups that were undernourished at a young age compared to groups who were normal or even already fat from childhood. We analyzed Hif-1α, Hif-2α, Lc3 mRNA by RT-qPCR; HIF-1α, HIF-2α, MAP1LC3A/LC3 protein level by ELISA. The HIF-1α and HIF-2α protein level of visceral adipocytes derived from the group of rat which were undernourished while young increased significantly compared to the group which was overnourished. The visceral adipocytes of the group which was overnourished since childhood showed an increase in Hif-2α mRNA level. The Lc3 mRNA of the rat group which were undernourished since young increased significantly compared to rat group which was obese since childhood.

Longer Hydroxyurea Administration Prior to Imatinib Mesylate is Risk Factor for Unsuccessful Major Molecular Response in Chronic-Phase Chronic Myeloid Leukemia: Possibility of P-Glycoprotein Role.

OBJECTIVE: This study aimed to identify the association between duration of HU administration prior to IM treatment and MMR achievement in chronic-phase CML while evaluating the role of MDA, HIF-1α and P-gp. METHODS: The study was conducted at Dr. Cipto Mangunkusumo National General Hospital and Dharmais Cancer Hospital, Jakarta using retrospective cohort design to analyse the association between the duration of HU before IM and its MMR achievement and cross-sectional design to analyse the association between MDA, HIF-1α and P-gp expressions with MMR achievement. Main subjects were chronic-phase CML patients treated by HU prior to IM for ≥ 12 months and HU only. The subjects were divided into four main groups: (1) chronic-phase CML patients treated with HU ≤ 6 months + IM ≥ 12 months and (2) HU > 6 months + IM ≥ 12 months (3) HU only (≤ 6 months), (4) HU only ( >6 months). Subjects were obtained from January 2015 to May 2016. Data were gathered through history taking, physical examination, medical record evaluation, and blood sample analysis. Bivariate analysis was conducted using chi square, independent T-test, and Mann-Whitney according to the variables. RESULTS: Administration of HU for more than 6 months prior to IM was associated with unsuccessful MMR achievement (RR 1.60; 95%CI 1.29-2.00). MDA level, HIF-1α, P-glycoprotein expression were not associated with MMR achievement but the mean MDA level (0.63±0.31 vs 0.75±0.41 p=0.461) and median P-glycoprotein expressions {16,92 (0,04 - 43,86) vs. 5,15 (0,02-39,64); p=0.311} were found to be higher in patients receiving HU for > 6 months group than in HU ≤ 6 months group consecutively. CONCLUSION: Administration of HU for more than 6 months prior to IM was associated with unsuccessful MMR achievement in chronic-phase CML. The study suggested that P-glycoprotein overexpression as the predictor for unsuccessful MMR achievement.

Expression and role of HIF-1α and HIF-2α in tissue regeneration: a study of hypoxia in house gecko tail regeneration.

The house gecko (Hemidactylus platyurus) has evolved the ability to autotomize its tail when threatened. The lost part is then regrown via epimorphic regeneration in a process that requires high energy and oxygen levels. Oxygen demand is therefore likely to outstrip supply and this can result in relative hypoxia in the tissues of the regenerating tail. The hypoxic state is stabilized by the Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α proteins. We induced tail autotomy in 30 mal H. platyurus adults using a standard procedure and then collected samples of the regenerated tail tissue on days 1, 3, 5, 8, 10, 13, 17, 21, 25, and 30 post autotomy. For each sample, mRNA expression was analyzed by qPCR, proteins were analyzed using Western Blot tests and immunohistochemistry, and the histological structure was analyzed using Hematoxylin and Eosin staining. On day 1, HIF-1α mRNA expression increased and the tissue was dominated by leucocyte and erythrocyte cells. HIF-1α mRNA expression peaked on day 3, at which time some cells were actively proliferating, migrating, and differentiating. At the same time as HIF-1α expression decreased, HIF-2α mRNA expression increased, as did overall cellular activity. HIF-2α expression increased more gradually but was present over a longer period of time than HIF-1α. We hypothesize that HIF-1α helps to initially stimulate the tissue regeneration process while HIF-2α functionally takes over the role of HIF-1α after HIF-1α succumbs to the oxygen conditions, but we suspect that both HIF-1α and HIF-2α play a role in overcoming the tissue's hypoxic state.

Role of Hypoxia Inducible Factor-1 Alpha (HIF-1α) in Cytoglobin Expression and Fibroblast Proliferation of Keloids.

BACKGROUND: Keloids are characterized by an overabundance of collagen deposition due to elevated activity and proliferation of fibroblasts, which lead to hypoxic conditions. Adaptation to these conditions is regulated by the transcription factor hypoxia inducible factor-1α (HIF-1α). Cytoglobin (Cygb), a reactive oxygen species scavenger, is a target gene of HIF-1α. In our previous study, we showed that Cygb expression in keloid tissue was correlated with HIF-1α expression. However, whether HIF-1α regulates Cygb expression and the proliferation of keloid fibroblasts remained unclear. Therefore, this study aimed to determine the role of HIF-1α in Cygb expression and fibroblast proliferation of keloids. METHODS: This was an in vitro study using a primary culture of keloid fibroblasts in which ibuprofen was used to inhibit HIF-1α expression. The expression of HIF-1α and Cygb mRNA were analyzed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) methods, and their protein levels were analyzed using an enzyme-linked immunosorbent assay (ELISA). Fibroblast proliferation was analyzed using a Trypan blue exclusion assay. RESULTS: Inhibition of HIF-1α by ibuprofen decreased Cygb mRNA expression but not in all the samples, followed by a decrease in the protein level of Cygb. There was a positive correlation between the HIF-1α protein and Cygb mRNA, probably due to the regulation of Cygb by HIF-1α at the mRNA level, but not the protein level. The proliferation of keloid fibroblasts was significantly decreased and positively correlated with the HIF-1α protein. CONCLUSION: HIF-1α regulates Cygb expression and fibroblast proliferation in keloids.

Bone Marrow Stem Cells Anti-liver Fibrosis Potency: Inhibition of Hepatic Stellate Cells Activity and Extracellular Matrix Deposition.

Transplantation of bone marrow derived stem cells (BMSCs) has been reported inhibits liver fibrosis. Several in vitro studies by co-culturing BMSCs and hepatic stellate cells (HSCs) indirectly or directly in 2D models showed inhibition of HSC as the key player in liver fibrosis. In this study, we investigated direct effect of BMSCs on HSCs by co-culturing BMSCs and HSCs in 3D model as it represents the liver microenvironment with intricate cell-cell and cell-matrix interactions. Primary isolated rat HSCs and BMSCs were directly co-cultured at 1:1 ratio with hanging drop method. The monoculture of rat HSCs served as positive control. Mono-culture and co-culture samples were harvested on day 3, 5 and 7 for histological analysis. The samples were analyzed for extracellular matrix deposition by Masson's Trichrome staining, tenascin-C immunocytochemistry, resting HSC's state as shown by positive Oil Red O stained cells. Our results indicated CD90＋CD34- BMSCs anti-liver fibrosis potency as evidenced by higher proportion of Oil Red O-positive cells in the co-culture group compared to the monoculture group and the significant decrease in extracellular matrix deposition as well as the decrease in tenascin-C expression in the co-culture group (p<0.05) compared to the monoculture group. These findings demonstrate that BMSCs have a potential therapeutic effect against liver fibrotic process through their capacity to inhibit HSCs activation and their effect in minimizing extracellular matrix deposition.