Assessment of resveratrol on diabetes of zebrafish (Danio rerio).

Purpose: Zebrafish (Danio rerio) is an established model for studying various metabolic diseases. The aim of this study was to evaluate the effect of resveratrol as a natural polyphenol on reducing inflammation caused by hyperglycemia (diabetes) and its effect on digestive tissue as well as TNF-α, IFN-γ, and INL1ß genes in zebrafish. Methods: Within a 20-day period, the research was performed on 120 adult zebrafish, which were randomly classified into eight groups: two experimental treatments (induced glucose = +G) and (without glucose = -G), where each main group was as follows: CTRL = control and RSV resveratrol with doses 10, 20, and 30 µmol/L. At the end of the period, the blood glucose level was measured using glucose test strip, staining of intestinal tissue was done by hematoxylin and eosin (H&E), and expression of INF-γ, IL1-ß, and TNF-α genes extracted from the intestinal was measured via internal method RT-PCR. Data analysis in this study was performed using SPSS software version 21. One-way ANOVA and mean comparison of treatments by Duncan test were used for data analysis. All statistical analyses were performed at a significant level (P < 0.5) where the mean data were presented with standard deviation. Results: According to the results, the lowest blood sugar level at the end of the experiment belonged to the group (G-RSV20) where no significant difference was observed between treatments (P > 0.05). The highest expression of IL1-ß gene belonged to the (G + CTRL) group (P < 0.05), while the (G + RSV20) group showed the lowest expression of the INF-γ gene and had a significant difference with other groups (P < 0.05). In (G + RSV10) treatment, the lowest expression of TNF-α gene was observed and there was no significant difference with other treatments (P > 0.05). Resveratrol would improve the absorption of nutrients in the intestinal tissue by increasing the number of goblet cells as well as the width and height of the villi. Conclusion: Collectively, this study indicated that treatment with resveratrol could improve metabolic-mediated performances by reducing blood glucose, increasing food absorption in the intestine tissue, and reducing the expression of inflammatory genes in type 2 diabetic zebrafish model.

Gut micobiota alteration by Lactobacillus rhamnosus reduces pro-inflammatory cytokines and glucose level in the adult model of Zebrafish.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is still a challenge for physicians to manage patient's circumstances. It is assumed that alterations in the normal flora may be involved in the pathogenesis of T2DM through inducing chronic inflammation. To investigate the effect of Lactobacillus rhamnosus as a common probiotic on T2DM, we induced an experimental model of T2DM in adult male Zebrafish by gradient hyper-glucose accumulation methodology. RESULTS: In this trial 3-month old male adult Zebrafish were divided in to four groups including two control groups and T2DM induced groups with or without probiotic treatment. After 5 days of acclimation, T2DM was induced by a gradient hyper-glucose accumulation methodology. Diabetic fishes had statistically abnormal blood glucose and pro-inflammatory cytokine levels compared to control group (p = 0.0001). These results suggest that probiotic intervention decreased the blood glucose level in the T2DM-P group by decreasing pro-inflammatory cytokines responsible for signaling in T2DM therapeutic modalities.

Correction to: Concurrent metformin and silibinin therapy in diabetes: assessments in zebrafish (Danio rerio) animal model.

[This corrects the article DOI: 10.1007/s40200-020-00637-7.].

Concurrent metformin and silibinin therapy in diabetes: assessments in zebrafish (Danio rerio) animal model.

OBJECTIVE: In this study, zebrafish was used as a biological model to induce type 2 diabetes mellitus through glucose. Then, the effect of metformin and silibinin combination was examined on elevated blood glucose, intestinal tissues, liver enzymes, and TNF-α, IFN-γ, INL1ß genes as inflammation marker genes. METHODS: The liver enzymes (AST, ALT, and ALP) derived from fish viscera homogenate supernatants were assayed in an auto-analyzer. The expression of target genes was quantified on RNA extracted from the tails by an in-house RT-PCR method, with fine intestine tissue staining performed by hematoxylin and eosin protocol (H&E). RESULT: In the glucose-free treatments, metformin and silymarin decreased the levels of AST, ALT, and ALP enzymes in the blood. The combination of these two drugs had also a significant role in reducing glucose levels. The body weight increased significantly in the control group which was affected by glucose concentration, with the lowest body weight gain observed in the metformin group. The expression of INL-1ß gene was significantly enhanced in the control group and the highest IFN-γ expression was observed in both control groups with glucose (G + CTRL) and without glucose (G-CTRL) (p < 0.05). The lowest level of TNF-α gene expression was observed in the control + glucose group (G + CTRL) (p < 0.05). Diabetic state causes weak absorption whereby the fish body demands to increase absorption level by enhancing the amount of acidic goblet cells thereby acidifying the environment in the gastric tracts. CONCLUSION: Collectively, this study indicated that treatment with metformin and Silibinin could improve metabolic-mediated performances by reducing the expression of inflammatory genes and blood glucose, modulating liver enzymes, and ameliorating the intestinal inflammation in type 2 diabetic zebrafish model.

Zebrafish as a Model Organism for the Development of Drugs for Skin Cancer.

Skin cancer, which includes melanoma and squamous cell carcinoma, represents the most common type of cutaneous malignancy worldwide, and its incidence is expected to rise in the near future. This condition derives from acquired genetic dysregulation of signaling pathways involved in the proliferation and apoptosis of skin cells. The development of animal models has allowed a better understanding of these pathomechanisms, with the possibility of carrying out toxicological screening and drug development. In particular, the zebrafish (Danio rerio) has been established as one of the most important model organisms for cancer research. This model is particularly suitable for live cell imaging and high-throughput drug screening in a large-scale fashion. Thanks to the recent advances in genome editing, such as the clustered regularly-interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) methodologies, the mechanisms associated with cancer development and progression, as well as drug resistance can be investigated and comprehended. With these unique tools, the zebrafish represents a powerful platform for skin cancer research in the development of target therapies. Here, we will review the advantages of using the zebrafish model for drug discovery and toxicological and phenotypical screening. We will focus in detail on the most recent progress in the field of zebrafish model generation for the study of melanoma and squamous cell carcinoma (SCC), including cancer cell injection and transgenic animal development. Moreover, we will report the latest compounds and small molecules under investigation in melanoma zebrafish models.

Carbonic anhydrase III: a neglected isozyme is stepping into the limelight.

CONTEXT: Carbonic anhydrase III (CA III) is a cytosolic enzyme which is known to be highly expressed in the skeletal muscle and has been recently linked to important roles in several physiological processes. OBJECTIVE: This review is focused on properties of CA III, including its distribution, function, structure and modulation of enzymatic activity by activators or inhibitors. We also provide some novel data on its expression in murine tissues. METHODS: In this article, the most relevant literature on CA III has been covered. New information on the distribution has been obtained by immunohistochemical staining and western blotting. RESULTS AND CONCLUSION: CA III shows the highest expression in the skeletal muscle and liver. Several other tissues contain lower levels of the enzyme. Activation or inhibition of CA III may offer a novel opportunity to treat some of the diseases linked to the defective expression or function of this enzyme.

Acetaldehyde-derived modifications on cytosolic human carbonic anhydrases.

Acetaldehyde can generate modifications in several proteins, such as carbonic anhydrase (CA) II. In this study, we extended in vitro investigations on acetaldehyde adduct formation by focusing on the other human cytosolic CA enzymes I, III, VII, and XIII. High-resolution mass spectrometric analysis indicated that acetaldehyde most efficiently formed covalent adducts with CA II and XIII. The binding of up to 19 acetaldehydes in CA II is probably attributable to the high number of lysine residues (n = 24) located mainly on the surface of the enzyme molecule. CA XIII formed more adducts (up to 25) than it contains lysine residues (n = 16) in its primary structure. Acetaldehyde treatment induced only minor changes in CA catalytic activity in most cases. The present study provides the first evidence that acetaldehyde can bind to several cytosolic CA isozymes. The functional consequences of such modifications will be further investigated in vivo by using animal models.

Novel carbonic anhydrase autoantibodies and renal manifestations in patients with primary Sjogren's syndrome.

OBJECTIVE: Anti-carbonic anhydrase II (anti-CA II) antibodies have been related to renal manifestations of primary SS (pSS), and animal studies have even suggested a pathogenic role for them. However, not all pSS patients with renal tubular acidosis (RTA) present with anti-CA II antibodies. Recently, several novel CA isoenzymes have been recognized and we aimed to investigate whether antibodies to these are associated with renal manifestations of pSS. METHODS: We examined anti-CA II antibodies as well as anti-CA I, VI, VII and XIII antibodies by ELISA tests in 74 pSS patients on whom detailed nephrological examinations had been performed and, as controls, in 56 subjects with sicca symptoms, but no pSS. RESULTS: The levels of anti-CA I, II, VI and VII antibodies were significantly higher in patients with pSS compared with subjects with sicca symptoms but no pSS. None of the anti-CA antibodies was associated with the presence of complete or incomplete RTA or proteinuria or urinary α1m excretion in patients with pSS. However, levels of anti-CA II, VI and XIII antibodies correlated significantly with urinary pH, and inversely with serum sodium concentrations. The degree of 24-h urinary protein excretion correlated weakly with levels of anti-CA VII antibodies. CONCLUSION: Not only antibodies to CA II, but also anti-CA VI and XIII antibodies seem to be associated with renal acidification capacity in patients with pSS.

Crystal structure of the C183S/C217S mutant of human CA VII in complex with acetazolamide.

Human carbonic anhydrase VII (hCA VII) is a cytosolic member of the alpha-CA family. This enzyme is mainly localized in a number of brain tissues such as the cortex, hippocampus and thalamus and has been noted for its contribution in generating neuronal excitation and seizures. Recently, it has been also proposed that hCA VII may be involved in the control of neuropathic pain, thus its inhibition may offer a new approach in designing pain killers useful for combating neuropathic pain. We report here the X-ray crystallographic structure of a mutated form of human CA VII in complex with acetazolamide, a classical sulfonamide inhibitor. These crystallographic studies provide important implications for the rational drug design of selective CA inhibitors with clinical applications.

Analysis of a shortened form of human carbonic anhydrase VII expressed in vitro compared to the full-length enzyme.

Carbonic anhydrase (CA) enzymes are expressed in all organs of the mammalian body where they participate in important physiological functions. CA VII is a cytosolic isozyme which may be expressed as two forms according to the recent GenBank data. We designed a present study to express and characterize the human CA VII forms: full-length CA VII and short form (predicted to lack 56 residues from the N-terminus). Reverse transcriptase PCR analysis revealed mRNAs for both CA VII forms in the human brain. These different forms were expressed as recombinant proteins to investigate their biochemical properties. The full-length CA VII was used to raise a polyclonal antiserum in a rabbit, and the antiserum was then employed in western blot analyses and immunohistochemistry of mouse tissues. Data from mass spectrometry and comparative modeling showed that CA VII protein contains a single intramolecular disulfide bridge (Cys-56 to Cys-180) which is lacking in the short form. The computer model suggested distinctly different folding for the different forms. The more exposed structure and the absence of the disulfide bridge in the short form could make this protein more susceptible to degradation. In fact, this was realized in several protein purification efforts in which the short form readily degraded during the experimental procedures. From these results, we conclude that the full-length CA VII is a predominant active form in human brain and also in other tissues. In addition to the brain, CA VII is expressed in several other organs including the stomach, duodenum, colon, liver, and skeletal muscle. The distribution pattern suggests multiple functions for CA VII in different organs.

Modification of carbonic anhydrase II with acetaldehyde, the first metabolite of ethanol, leads to decreased enzyme activity.

BACKGROUND: Acetaldehyde, the first metabolite of ethanol, can generate covalent modifications of proteins and cellular constituents. However, functional consequences of such modification remain poorly defined. In the present study, we examined acetaldehyde reaction with human carbonic anhydrase (CA) isozyme II, which has several features that make it a suitable target protein: It is widely expressed, its enzymatic activity can be monitored, its structural and catalytic properties are known, and it contains 24 lysine residues, which are accessible sites for aldehyde reaction. RESULTS: Acetaldehyde treatment in the absence and presence of a reducing agent (NaBH3(CN)) caused shifts in the pI values of CA II. SDS-PAGE indicated a shift toward a slightly higher molecular mass. High-resolution mass spectra of CA II, measured with and without NaBH3(CN), indicated the presence of an unmodified protein, as expected. Mass spectra of CA II treated with acetaldehyde revealed a modified protein form (+26 Da), consistent with a "Schiff base" formation between acetaldehyde and one of the primary NH2 groups (e.g., in lysine side chain) in the protein structure. This reaction was highly specific, given the relative abundance of over 90% of the modified protein. In reducing conditions, each CA II molecule had reacted with 9-19 (14 on average) acetaldehyde molecules (+28 Da), consistent with further reduction of the "Schiff bases" to substituted amines (N-ethyllysine residues). The acetaldehyde-modified protein showed decreased CA enzymatic activity. CONCLUSION: The acetaldehyde-derived modifications in CA II molecule may have physiological consequences in alcoholic patients.