Comprehensive machine learning models for predicting therapeutic targets in type 2 diabetes utilizing molecular and biochemical features in rats.

Introduction: With the increasing prevalence of type 2 diabetes mellitus (T2DM), there is an urgent need to discover effective therapeutic targets for this complex condition. Coding and non-coding RNAs, with traditional biochemical parameters, have shown promise as viable targets for therapy. Machine learning (ML) techniques have emerged as powerful tools for predicting drug responses. Method: In this study, we developed an ML-based model to identify the most influential features for drug response in the treatment of type 2 diabetes using three medicinal plant-based drugs (Rosavin, Caffeic acid, and Isorhamnetin), and a probiotics drug (Z-biotic), at different doses. A hundred rats were randomly assigned to ten groups, including a normal group, a streptozotocin-induced diabetic group, and eight treated groups. Serum samples were collected for biochemical analysis, while liver tissues (L) and adipose tissues (A) underwent histopathological examination and molecular biomarker extraction using quantitative PCR. Utilizing five machine learning algorithms, we integrated 32 molecular features and 12 biochemical features to select the most predictive targets for each model and the combined model. Results and discussion: Our results indicated that high doses of the selected drugs effectively mitigated liver inflammation, reduced insulin resistance, and improved lipid profiles and renal function biomarkers. The machine learning model identified 13 molecular features, 10 biochemical features, and 20 combined features with an accuracy of 80% and AUC (0.894, 0.93, and 0.896), respectively. This study presents an ML model that accurately identifies effective therapeutic targets implicated in the molecular pathways associated with T2DM pathogenesis.

A neuroprotective effect of pentoxifylline in rats with diabetic neuropathy: Mitigation of inflammatory and vascular alterations.

BACKGROUND: Treatment of diabetic neuropathic pain does not change the natural history of neuropathy. Improved glycemic control is the recommended treatment in these cases, given that no specific treatment for the underlying nerve damage is available, so far. In the present study, the potential neuroprotective effect of pentoxifylline in streptozotocin (50 mg/kg) induced diabetic neuropathy in rats was investigated. METHODS: Pentoxifylline was administered at doses equivalent to 50, 100 & 200 mg/kg, in drinking water, starting one week after streptozotocin injection and for 7 weeks. Mechanical allodynia, body weight and blood glucose level were assessed weekly. Epidermal thickness of the footpad skin, and neuroinflammation and vascular alterations markers were assessed. RESULTS: Tactile allodynia was less in rats that received pentoxifylline at doses of 100 and 200 mg/kg (60 % mechanical threshold increased by 48 % and 60 %, respectively). The decrease in epidermal thickness of footpad skin was almost completely prevented by the same doses. This was associated with a decrease in spinal tumor necrosis factor alpha (TNFα) and nuclear factor kappa B levels and a decrease in microglial ionized calcium binding adaptor molecule 1 immunoreactivity, compared to the control diabetic group. In sciatic nerve, there was decrease in TNF-α and vascular endothelial growth factor levels and intercellular adhesion molecule immunoreactivity. CONCLUSION: Pentoxifylline showed a neuroprotective effect in streptozotocin-induced diabetic neuropathy, which was associated with a suppression of both the inflammatory and vascular pathogenic pathways that was not associated with a hypoglycemic effect. Thus, it may represent a potential neuroprotective drug for diabetics.

A comparative study between angiotensin receptor neprilysin inhibitor (thiorphan/irbesartan) with each of nitrate and carvedilol in a rat model of myocardial ischemic reperfusion injury.

The combined angiotensin receptor neprilysin inhibitor is a promising cardioprotective pharmacological agent. This study investigated the beneficial effects of thiorphan (TH)/irbesartan (IRB), in myocardial ischemia-reperfusion (IR) injury, compared to each of nitroglycerin and carvedilol. Male Wistar rats were divided into five groups (10 rats/group): Sham, untreated I/R, TH/IRB + IR (0.1/10 mg/kg), nitroglycerin + IR (0.2 mg/kg), and carvedilol + IR (10 mg/kg). Mean arterial blood pressure, cardiac functions and arrhythmia incidence, duration and score were assessed. Cardiac levels of creatine kinase-MB (CK-MB), oxidative stress, endothelin-1, ATP, Na+ /K+ ATPase pump activity and mitochondria complexes activities were measured. Histopathological examination, Bcl/Bax immunohistochemistry studies and electron microscopy examination of left ventricle were performed. TH/IRB preserved the cardiac functions and mitochondrial complexes activities, mitigated cardiac damage, reduced oxidative stress and arrhythmia severity, improved the histopathological changes and decreased cardiac apoptosis. TH/IRB showed a comparable effect to each of nitroglycerin and carvedilol in alleviating the IR injury consequences. TH/IRB showed significant preservation of mitochondrial complexes activity I and II compared to nitroglycerin. TH/IRB significantly increased LVdP/dtmax and decreased oxidative stress, cardiac damage and endothelin-1 along with increasing the ATP content, Na+ /K+ ATPase pump activity and mitochondrial complexes activity when compared to carvedilol. TH/IRB showed a cardioprotective effect in reducing IR injury that is comparable to each of nitroglycerin and carvedilol that could be explained in part by its ability to preserve mitochondrial function, increase ATP, decrease oxidative stress as well as endothelin 1.

Cyanidin-3-Glucoside Modulates hsa_circ_0001345/miRNA106b/ATG16L1 Axis Expression as a Potential Protective Mechanism against Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common form of malignancy in the liver. Autophagy was found to have a significant effect in controlling HCC. Anthocyanins, which are naturally occurring pigments in a variety of fruits and vegetables, have been thoroughly documented to be involved in a variety of bioactive activities and are widely employed for their antioxidant capabilities. Cyanidin-3-glucoside (C3G) extracted from Morus alba L. has promising antioxidant and anti-tumour activities. The current study aims to examine the protective action of C3G against hepatocellular carcinoma through the investigation of the autophagy protein ATG16L1 expression along with its related RNA molecules (hsa_circ_0001345 and miRNA106b) in Wistar rats. In vivo precancerous lesions (PCL) were induced using diethylnitrosamine (DEN) and acetamidofluorene (2-AAF). Rats were treated with C3G (10, 15, and 20 mg/kg; 4 times weekly) for 112 days (16 weeks). Liver function tests, alfa fetoprotein, ATG16L1 expression, hsa_circ_0001345, and miRNA106b differential expression were examined. Liver sections were examined by histological and immunohistochemical approaches. The current study's findings indicated that C3G administration protects against the negative effects of DEN-2-AAF on liver functions and liver histopathological sections, which nominated C3G as a potential prophylactic agent against HCC.

Promotive action of 2-acetylaminofluorene on hepatic precancerous lesions initiated by diethylnitrosamine in rats: Molecular study.

BACKGROUND: Diethylnitrosamine (DEN) induces hepatic neoplastic lesions over a prolonged period. AIM: To investigate the promotive action of 2-acetylaminofluorene (2-AAF) when combined with DEN in order to develop a rat model for induction of precancerous lesion and investigate the molecular mechanism underlying the activity of 2-AAF. METHODS: The pre-precancerous lesions were initiated by intraperitoneal injection of DEN for three weeks consecutively, followed by one intraperitoneal injection of 2-AAF at three different doses (100, 200 and 300 mg/kg). Rats were separated into naïve, DEN, DEN + 100 mg 2-AAF, DEN + 200 mg 2-AAF, and DEN + 300 mg 2-AAF groups. Rats were sacrificed after 10 wk and 16 wk. Liver functions, level of alpha-fetoprotein, glutathione S-transferase-P and proliferating cell nuclear antigen staining of liver tissues were performed. The mRNA level of RAB11A, BAX, p53, and Cyclin E and epigenetic regulation by long-noncoding RNA (lncRNA) RP11-513I15.6, miR-1262 (microRNA), and miR-1298 were assessed in the sera and liver tissues of the rats. RESULTS: 2-AAF administration significantly increased the percent area of the precancerous foci and cell proliferation along with a significant decrease in RAB11A, BAX, and p53 mRNA, and the increase in Cyclin E mRNA was associated with a marked decrease in lncRNA RP11-513I15.6 expression with a significant increase in both miR-1262 and miR-1298. CONCLUSION: 2-AFF promoted hepatic precancerous lesions initiated through DEN by decreasing autophagy, apoptosis, and tumor suppression genes, along with increased cell proliferation, in a time- and dose-dependent manner. These actions were mediated under the epigenetic regulation of lncRNA RP11-513I15.6/miR-1262/miR-1298.

Cyanidin 3-glucoside modulated cell cycle progression in liver precancerous lesion, in vivo study.

BACKGROUND: Cyanidin-3-O-glucoside (cyan) exhibits antioxidant and anticancer properties. The cell cycle proteins and antimitotic drugs might be promising therapeutic targets in hepatocellular carcinoma. AIM: To investigate the effect of cyan administration on cell cycle in hepatic precancerous lesion (PCL) induced by diethylnitrosamine/2-acetylaminofluorene (DEN/2-AAF) in Wistar rats. METHODS: In vivo, DEN/2-AAF-induced hepatic PCL, rats were treated with three doses of cyan (10, 15, and 20 mg/kg/d, for four consecutive days per week for 16 wk). Blood and liver tissue samples were collected for measurement of the followings; alpha fetoprotein (AFP) liver function and RNA panel differential expression was evaluated via real time polymerase chain reaction. Histopathological examination of liver sections stained with H&E and immunohistochemical study using glutathione S-transferase placental (GSTP) and proliferating cell nuclear antigen (PCNA) antibodies were assessed. RESULTS: Cyan administration mitigated the effect of DEN/2-AFF induced PCL, decreased AFP levels, and improved liver function. Remarkably, treatment with cyan dose dependently decreased the long non-coding RNA MALAT1 and tubulin gamma 1 mRNA expressions and increased the levels of miR-125b, all of which are involved in cell cycle and mitotic spindle assembly. Of note, cyan decreased GSTP foci percent area and PCNA positively stained nuclei. CONCLUSION: Our results indicated that cyan could be used as a potential therapeutic agent to inhibit liver carcinogenesis in rat model via modulation of cell cycle.

Epigenetic modulation of autophagy genes linked to diabetic nephropathy by administration of isorhamnetin in Type 2 diabetes mellitus rats.

Aim: To assess isorhamnetin efficacy for diabetic kidney disease in a Type 2 diabetes mellitus rat model, through investigating its effect at the epigenetic, mRNA and protein levels. Materials & methods: Type 2 diabetes mellitus was induced in rats by streptozotocin and high-fat diet. Rats were treated with isorhamnetin (50 mg/kg/d) for 4 or 8 weeks. Fasting blood glucose, renal and lipid profiles were evaluated. Renal tissues were examined by light and electron microscopy. Autophagy genes (FYCO1, ULK, TECPR1 and WIPI2) and miR-15b, miR-34a and miR-633 were assessed by qRT-PCR, and LC3A/B by immunoblotting. Results: Isorhamnetin improved fasting blood glucose, renal and lipid profiles with increased autophagosomes in renal tissues. It suppressed miRNA regulation of autophagy genes. Conclusion: We propose a molecular mechanism for the isorhamnetin renoprotective effect by modulation of autophagy epigenetic regulators.

Exosomal miR-1298 and lncRNA-RP11-583F2.2 Expression in Hepato-cellular Carcinoma.

AIM: The aim of this study was to explore the expression of exosomal non-coding RNAs (ncRNAs) in the sera of patients with HCC versus control. METHODS: Firstly, Bioinformatics analysis was conducted to retrieve ncRNAs specific to HCC (hsa-miRNA-1298 and lncRNA-RP11-583F2.2). Afterwards, extraction and characterization of exosomes were performed. We measured the expression of the chosen exosomal RNAs by reverse transcriptase quantitative real-time PCR in sera of 60 patients with HCC, 42 patients with chronic hepatitis C (CHC) infection and 18 healthy normal volunteers. RESULTS: The exosomal ncRNAs [hsa-miRNA-1298, lncRNA-RP11-583F2.2] had better sensitivity and specificity than alpha-fetoprotein (AFP) in HCC diagnosis. CONCLUSION: The exosomal hsa-miRNA-1298, lncRNA-RP11-583F2.2 can be potential biomarkers for HCC diagnosis.

Pentoxifylline alleviated cardiac injury via modulating the cardiac expression of lncRNA-00654-miR-133a-SOX5 mRNA in the rat model of ischemia-reperfusion.

Pentoxifylline (PTX) protects from many cardiovascular complications. It plays a critical role in stem cell proliferation and differentiation. Here, the effect of PTX administration on cardiac ischemia and dysfunction was explored. PTX in 3 doses (20, 30, and 40 mg/kg), was administered in vivo 5 min before a 45 min occlusion of the left anterior descending artery, followed by a 120 min reperfusion in male Wistar rats. The left ventricular end-diastolic pressure and dP/dtmax were assessed. Blood and cardiac tissue samples were collected for measuring the levels of cardiac enzymes and the expression of lncRNA-00654-miR-133a-SOX5. Samples of left ventricles were collected and processed for light microscopic, immunohistochemical staining for c-kit (a marker for cardiac progenitor cells) and transmission electron microscopic examination. PTX administration showed improvements in cardiac function tests, enzymes, and myocytes. Microscopic features showed minimal cardiac edema, hemorrhage, cellular inflammatory infiltration and fibrosis in addition to increased c-kit + cells in cardiac tissue samples. Notably, this treatment also produced a dose-dependent decrease in lncRNA-00654 with an increase in SOX5 mRNA and miRNA-133a-3p expressions. In conclusion, PTX has the potential to alleviate cardiac injury and increase the number of c-kit + cells following ischemia-reperfusion in the rat model via modulation of lncRNA-00654 and miR-133a-SOX5 mRNA expressions.

N-acetylcysteine (NAC) alleviates the peripheral neuropathy associated with liver cirrhosis via modulation of neural MEG3/PAR2/ NF-Ò¡B axis.

BACKGROUND AND AIM: Oxidative stress (OS) is accused in pathogenesis of many diseases, including liver cirrhosis by many mechanisms. One of them is the disturbance of long non coding maternally expressed 3 (MEG3)/protease activated receptor 2 (PAR2) downstream pathway. We aimed to investigate the role of this axis in cirrhotic neuropathy and whether an antioxidant compound such as N-acetylcysteine (NAC) could improve the peripheral nerve function through repression of MEG3/PAR2. METHODS: Thirty Wistar rats were used and divided into 5 groups; naive, thiacetamide (TAA) (200 mg/kg 3 times/week. i.p. for 8 weeks) and TAA+NAC (50 or 100 or 200 mg/kg/day) groups. Von Frey (VF) test for mechanical nociceptive responses, hepatic& neural MEG3, NF-Ò¡B and neural PAR2 expression by PCR, histological studies for liver and sciatic nerve together with the dorsopedal skin thickness were done. RESULTS: TAA induced significant decrease in liver function, negative VF test, an increase in the expression of hepatic& neural MEG3, NF-Ò¡B and neural PAR2. The histological studies showed cirrhotic changes with atrophy of the sciatic nerve and the dorsal skin. NAC improved the liver function together with reversal of the neural: functional, biochemical and histological changes in a dose dependent manner. CONCLUSIONS: NAC could improve the peripheral neuropathy in cirrhotic rat through suppression of MEG3/PAR2 expression.

Potential protective effects of Dapagliflozin in gentamicin induced nephrotoxicity rat model via modulation of apoptosis associated miRNAs.

BACKGROUND: Drug-induced kidney injury (DIKI) can be manifested with progressive chronic kidney diseases or end-stage renal diseases. Understanding the molecular disarrangements caused by DIKI is an attractive point of interest. A class of non-coding RNA called microRNAs (miRNAs) is known to play a major role in regulation of gene expression and signaling pathways making miRNAs excellent targets for new therapeutic agents. AIM OF THE STUDY: We aimed to investigate the role of miRNA 21 and 181a in gentamicin (GNT) induced nephrotoxicity rat model and the protective effect of Dapagliflozin (DAPA) in modulating their expression through studying its effect on renal function as well as renal histopathological changes. MATERIALS AND METHODS: Wistar rats were used and divided into: naïve, DAPA, GNT and DAPA + GNT groups. In all studied groups, kidney function, oxidative stress, apoptosis markers and miRNAs' expression in serum and renal biopsies were investigated in addition to the histopathological studies to identify its early renoprotective effect. RESULTS: DAPA was found to improve kidney function, oxidative stress markers, decrease apoptosis of renal tubular cells and increase miR-21 but decrease the expression of miR-181a with restoration of the renal architecture after 14 days of treatment in GNT induced nephrotoxicity rat model. CONCLUSIONS: DAPA produced significant decrease in renal expression of miR-181a on the other hand it increased the expression of renal miR-21, this may introduce a novel early protective effect of DAPA against GNT-induced nephrotoxicity.

Pantoprazole attenuates tumorigenesis via inhibition of exosomal secretion in a rat model of hepatic precancerous lesion induced by diethylnitrosamine and 2-acetamidofluorene.

The present study aimed to evaluate the potential therapeutic effect of pantoprazole, a proton-pump inhibitor, on precancerous lesion (PCL) in rats. diethylnitrosamine and 2-acetylaminofluorene were used to induce PCL in rats, in vivo. The rats were treated with three doses of pantoprazole (100, 50, and 25  mg/kg; three times weekly) during the last 4 weeks of the total 10 weeks of the experiment. Blood and liver tissue samples were collected for measurement of the exosomal abundance and exosomal competing endogenous RNA markers. Results revealed that pantoprazole administration had an ameliorating effect on liver function tests and microscopic features of the liver; and decreased exosome abundance in the liver tissue samples and sera of the rats. Meanwhile, the treatment also resulted in a dose-dependent decrease in exosomal RAB11A mRNA and long noncoding RNA RP11-513I15.6, which is an important participant in th exosomal secretion process with an increase in exosomal miRNA-1262. Based on these results, we postulated that pantoprazole has the potential to attenuate liver tumorigenesis in this rat model.

Coenzyme Q10 attenuates lung and liver fibrosis via modulation of autophagy in methotrexate treated rat.

The present study was conducted to investigate the potential protective effects of coenzyme Q 10 (CoQ10) administration on methotrexate induced lung and liver fibrosis in rat model, and to explore our hypothesis regarding its possible mechanism of action through reactivation of autophagy pathway. Methotrexate induced fibrosis was achieved by intraperitoneal injections twice a week for 4 weeks. A combined treatment of CoQ10 and methotrexate were used. Blood samples for biochemical analysis, lung and livers tissue for biochemical and histopathological analysis, were investigated. Concomitant treatment of CoQ10 & methotrexate caused improvement in histological picture of the lung and liver tissues, liver function and oxidative stress biomarkers, modulation of autophagy genes [mammalian target of rapamycin (m-TOR), Microtubule-associated proteins 1 A/1B light chain 3 (MAP1LC3B), and Sequestosome 1 ubiquitin-binding protein p62 (p62/SQSTM1)] with simultaneous reduction in High Mobility Group Protein B1 (HMGB1). Based on our results we postulated that CoQ10 up regulates autophagy pathway that could explain its protective properties against lung and liver fibrosis caused by methotrexate treatment in current study rat model.

Role of exosomal competing endogenous RNA in patients with hepatocellular carcinoma.

Recent research has tried to use exosomal RNAs (coding and noncoding) as potential diagnostic markers for hepatocellular carcinoma (HCC). Initially, by using bioinformatics, we selected an HCC-exosomal RNA-based biomarker panel. The choice of this panel depends on the integration of Ras-related in brain (RAB11A) gene expression and its competing endogenous network. This network includes long noncoding RNA RP11-513I15.6 (lncRNA-RP11-513I15.6) and microRNA-1262 (miR-1262). Secondly, we tried to validate the expression of this network in the sera of 60 patients with HCC in comparison with 42 chronic hepatitis C virus-infected patients and 18 healthy controls. Then we assessed the diagnostic efficiency of this panel using a receiver operating characteristic curve analysis. The panel of 3 exosomal RNA-based biomarkers (lncRNA-RP11-513I15.6, miR-1262, and RAB11A) showed excellent sensitivity and specificity in discriminating patients with HCC from patients with chronic hepatitis C virus and healthy controls. Among these 3 RNAs, serum RAB11A mRNA was the most independent prognostic factor. The selected circulatory exosomal RNA-based biomarker panel showed its ability to be used as a diagnostic and prognostic biomarker tool for HCC. Moreover, these biomarkers could be therapeutic targets.

Hyperglycemia Alters the Protein Levels of Prominin-1 and VEGFA in the Retina of Albino Rats.

In this study, we addressed the potential relationship between prominin-1 (prom1) and vascular endothelial growth factor (VEGFA) in diabetes-induced retinopathy. In total, we examined 28 retinas from 14 rats with streptozotocin-induced diabetes and 30 retinas from 15 untreated control rats. ELISA was used to measure the level of prom1 and VEGFA in retinal tissue homogenates. Immunohistochemical techniques were used with antibodies directed against prom1, VEGFA, and CASP-3. After 180 days of diabetes induction, we performed light and electron microscopy studies on rat eyes to evaluate histopathological changes and to estimate the de novo metric "Diabetic Retinopathy Histopathological Index" (DRHI). These changes were then correlated to the tissue and immunoexpression levels of prom1 and VEGFA. The data showed a significant upregulation of the tissue levels and optical densities (ODs) of VEGFA and prom1 immunoreactivity in diabetic retinas compared with controls. Both the tissue levels and OD values of prom1 and VEGFA correlated significantly with each other and to the diabetic structural changes as calculated by DRHI. Taken together, these data provide new insight into the potential role of prom1 and VEGFA in the development of diabetic retinopathy.

Caffeic Acid Attenuates Diabetic Kidney Disease via Modulation of Autophagy in a High-Fat Diet/Streptozotocin- Induced Diabetic Rat.

The aim of this study is to evaluate the anti-diabetic nephropathy effect of Caffeic acid and to prove our hypothesis for its mechanism of action that it may occur by reactivation of autophagy pathway via suppression of autophagy regulatory miRNAs. In vivo, high-fat diet and streptozotocin-induced (HFD-STZ) diabetic rats were treated with Caffeic acid once per day for 12 weeks before and after development of diabetic nephropathy. Blood and urine biochemical parameters, autophagy transcripts and their epigenetic regulators together with renal tissue morphology were investigated. In diabetic rats, Caffeic acid intake, caused improvement in albumin excretion,blood glucose, reduced renal mesangial matrix extension with increased vacuolation and reappearance of autophagosomes. Meanwhile, it resulted in autophagy genes up-regulation [RB 1-inducible coiled coil protein (RB1CC1), Microtubule-associated proteins 1A/1B light chain 3(MAP1LC3B), Autophagy related gene (ATG-12),] with simultaneous reduction in their epigenetic regulators; miRNA-133b, -342 and 30a, respectively. These above mentioned effects were more significant in the diabetic nephropathy Caffeic treated rats than in the prophylactic group. Based on our results we postulated that caffeic acid modulates autophagy pathway through inhibition of autophagy regulatory miRNAs, that could explain its curative properties against diabetic kidney disease.

Safety of Intracameral Injection of Minimal Bactericidal Concentration of Povidone Iodine on the Corneal Endothelium in a Rabbit Model.

PURPOSE: To investigate the safety of intracameral injection of minimum bactericidal concentration (MBC) of povidone iodine (PI) on the corneal endothelium in a rabbit model as a proposed method of prophylaxis against postoperative endophthalmitis. METHODS: We included 32 New Zealand white rabbits in the study. Twenty-four rabbits received intracameral injections of 0.1 mL of 0.25% PI, and they were sequentially killed at intervals; first, seventh, and 14th day. The control group included 4 rabbits that received intracameral injections of 0.1 mL normal saline, and 4 rabbits that underwent the same intraocular procedure without injections (sham operated). Slit-lamp examination and ultrasonic corneal pachymetry were performed before and after injections for both eyes. The corneas were histopathologically examined by light and electron microscopy. RESULTS: MBC of PI (0.25%) was toxic to rabbits' corneal endothelium as evident by histopathological changes, corneal edema, and increased corneal thickness on day 1. Signs of healing were obvious on day 7 and were almost complete on day 14, as detected by histopathology, subsidence of corneal edema, and normalization of corneal thickness. CONCLUSIONS: MBC (0.25%) of PI was found toxic to the rabbits' corneal endothelium, with progressive regeneration and complete healing within 2 weeks. To our knowledge, we are the first to use MBC of PI in intracameral injection trials. Further studies on primates, which have more comparable regenerative capacity to humans' corneal endothelium, are encouraged to evaluate their endothelial healing response.