Immunohistochemical Staining For Caspase-3 Research Articles

Age changes in the gastric mucosa of male albino rat: histological, immunohistochemical, histomorphometric and biochemical study.

Age related changes in the stomach are associated with alterations in the structure and secretory function of the gastric glands. The present study aimed to investigate histological, histomorphometric and biochemical changes in the gastric mucosa of rats with age. Eighty adult male albino rats were randomly divided into four age groups, 20 rats in each (prepubertal, adolescent, adult, and senile). The gastric specimens were subjected to light microscopic examination using haematoxylin and eosin, PAS and Masson's trichrome stains. Immunohistochemical staining for caspase-3 and inducible nitric oxide synthase (iNOS) was carried out. Measurement of superoxide dismutase (SOD), glutathione peroxidase (GPx) and malondialdehyde (MDA) activity in gastric tissue homogenates was performed using ELISA. Quantitative analysis of vascular endothelial growth factor (VEGF) gene expression was done by real-time polymerase chain reaction (PCR). Light microscopic examination of gastric mucosa of senile rats revealed distortion of gastric glands and erosions. Surface mucous cells, mucous neck cells, parietal and chief cells exhibited cytoplasmic destruction, nuclear degeneration, apoptosis and oxidative damage. There was a significant decrease in the mean gastric mucosal thickness, increase in collagen content and decrease in mucous content with the advance of age. These morphological changes were associated with a significant decrease in SOD and GPx activity and increase in MDA activity, in addition to decreased VEGF gene expression. Gastric mucosa of aged rats showed histological and immunohistochemical alterations. These changes were associated with oxidative stress, decreased antioxidant capacity and decreased angiogenesis.

Investigating neuropathological changes and underlying neurobiological mechanisms in the early stages of primary blast-induced traumatic brain injury: Insights from a rat model

The utilization of explosives and chemicals has resulted in a rise in blast-induced traumatic brain injury (bTBI) in recent times. However, there is a dearth of diagnostic biomarkers and therapeutic targets for bTBI due to a limited understanding of biological mechanisms, particularly in the early stages. The objective of this study was to examine the early neuropathological characteristics and underlying biological mechanisms of primary bTBI. A total of 83 Sprague Dawley rats were employed, with their heads subjected to a blast shockwave of peak overpressure ranging from 172 to 421 kPa in the GI, GII, and GIII groups within a closed shock tube, while the body was shielded. Neuromotor dysfunctions, morphological changes, and neuropathological alterations were detected through modified neurologic severity scores, brain water content analysis, MRI scans, histological, TUNEL, and caspase-3 immunohistochemical staining. In addition, label-free quantitative (LFQ)-proteomics was utilized to investigate the biological mechanisms associated with the observed neuropathology. Notably, no evident damage was discernible in the GII and GI groups, whereas mild brain injury was observed in the GIII group. Neuropathological features of bTBI were characterized by morphologic changes, including neuronal injury and apoptosis, cerebral edema, and cerebrovascular injury in the shockwave's path. Subsequently, 3153 proteins were identified and quantified in the GIII group, with subsequent enriched neurological responses consistent with pathological findings. Further analysis revealed that signaling pathways such as relaxin signaling, hippo signaling, gap junction, chemokine signaling, and sphingolipid signaling, as well as hub proteins including Prkacb, Adcy5, and various G-protein subunits (Gnai2, Gnai3, Gnao1, Gnb1, Gnb2, Gnb4, and Gnb5), were closely associated with the observed neuropathology. The expression of hub proteins was confirmed via Western blotting. Accordingly, this study proposes signaling pathways and key proteins that exhibit sensitivity to brain injury and are correlated with the early pathologies of bTBI. Furthermore, it highlights the significance of G-protein subunits in bTBI pathophysiology, thereby establishing a theoretical foundation for early diagnosis and treatment strategies for primary bTBI.

Naringin Inhibits Colorectal Carcinogenesis by Inhibiting Viability of Colorectal Cancer Cells.

To explore the therapeutic effect of naringin on colorectal cancer (CRC) and the related mechanism. Cell counting kit-8 (CCK-8) assay and annexin V-FITC/PI assay were used to detect the effect of naringin (50-400 µg/mL) on cell proliferation and apoptosis of CRC cells, respectively. The scratch wound assay and transwell migration assay were used to assess the effect of naringin on CRC cell migration. Four-week-old male nude mice were injected with HCT116 cells subcutaneously to establish the tumor xenograft model. Naringin was injected intraperitoneally at 50 mg/(kg·d), with solvent and 5-fluorouracil treatment as control. The width and length of the tumors were measured and recorded every 6 days, and tumor tissues were photographed and weighed on the last day of the 24-d observation period. Immunohistochemical staining for caspase-3, proliferating cell nuclear antigen and TUNEL assay were used to evaluate the effect of naringin on cell proliferation and apoptosis in tumor tissues. The body weight, food and water intake of mice were recorded, and the major organs in different treatment groups were weighed on the last day and stained with hematoxylin and eosin for histological analysis. Meanwhile, the routine blood indicators were recorded. CCK-8 and annexin V-FITC/PI results confirmed that naringin (100, 200, and 400 µg/mL) could inhibit proliferation and promote apoptosis. The scratch wound assay and transwell migration assay results confirmed the inhibitory activity of naringin against CRC cells migration. In vivo results demonstrated the inhibitory effect of naringin on tumor growth with good bio-compatibility. Naringin inhibited colorectal carcinogenesis by inhibiting viability of CRC cells.

Stabilization of glutathione redox dynamics and CYP2E1 by green synthesized Moringa oleifera-mediated zinc oxide nanoparticles against acrylamide induced hepatotoxicity in rat model: Morphometric and molecular perspectives.

The terrible reality is that acrylamide (AA) is a common food contaminant found in a wide variety of commonly consumed foods. This research involves the advancement of a more dependable technique for the bio-fabrication of zinc oxide nanoparticles (ZNPs) through the green method using Moringa Oleifera extract (MO-ZNPs) as an efficient chelating agent for acrylamide (AA). The effects of AA on glutathione redox dynamics, liver function, lipid profile, and zinc residues in Sprague Dawley rats are investigated. Finally, the microarchitecture and immunohistochemical staining of Caspase-3 and CYP2E1 were determined in the liver tissue of rats. Four separate groups, including control, MO-ZNPs (10mg/kg b. wt), AA (20mg/kg b. wt), and AA+MO-ZNPs for 60 days. The results revealed a suppressed activity of glutathione redox enzymes (GSH, GPX,and GSR) on both molecular and biochemical levels. Also, AA caused elevated liver enzymes, hepatosomatic index, and immunohistochemical staining of caspase-3 and CYP2E1 expression. MO-ZNPs co-treatment, on the other hand, stabilized glutathione-related enzyme gene expression, normalized hepatocellular enzyme levels, and restored hepatic tissue microarchitectures. It could be assumed that MO-ZNPs is a promising hepatoprotective molecule for alleviating AA-induced hepatotoxicity. We witnessed changes in glutathione redox dynamics to be restorative. Glutathione and cytochrome P450 2E1 play crucial roles in AA detoxification, so maintaining a healthy glutathione redox cycle is necessary for disposing of AA toxicity.

Cytotoxicity associated with acute and chronic administration of synthetic cannabinoids “Strox” in the brain, liver, heart, and testes of male albino rats: histological and immunohistochemical study

BackgroundSynthetic cannabinoids are one of the largest groups of new psychoactive substances that invaded Egypt’s drug abuse market over the past few years.AimRandomized controlled trial study to demonstrate the effects of acute and chronic toxicity by synthetic cannabinoid (Strox) on the brain, liver, heart, and testes in adult male albino rats through histopathological examination by light microscope and immunohistochemistry.MethodsTotal number of fifty male albino rats were divided into five different groups, two control and three treated groups. Negative and positive control groups received distilled water and dimethyl sulfoxide, respectively, acute group received LD50 (lethal dose 50) once and observed for 14 days, chronic group received 1/10 LD50 for 3 months, and finally chronic withdrawal groups received 1/10 LD50 for 3 months and then left 2 weeks without the substance to observe the withdrawal manifestations.ResultsThe current study revealed various histopathological changes in all organs with increased expression of cannabinoid receptor 1. The most important findings observed by light microscope examination were shrinkage and degenerative changes in Purkinje cells in brain sections, abnormalities in blood sinusoids and architecture in liver section, disruption in cardiac muscle fiber in heart sections, and finally testes showed irregularities in seminiferous tubules and germinal cells. Immunohistochemical staining for caspase-3 in the brain, liver, and heart showed weak-positive reaction in acute group and a strong reaction with chronic groups. Additionally, increase in collagen fiber was observed in sections of the liver and heart of chronic group.ConclusionsSynthetic cannabinoid sample (Strox) toxicity caused adverse effects on the brain, liver, heart, and testes as shown by increasing cannabinoid receptor 1 and caspase-3 expression.

Spontaneous Murine Model of Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the carcinogenesis and development of ATC, as well as therapeutic methods, since standard treatments are essentially depleted in ATC patients. However, low prevalence has hampered thorough clinical studies and the collection of tissue samples, so little progress has been achieved in creating effective treatments. We used genetic engineering to create a conditionally inducible ATC murine model (mATC) in a C57BL/6 background. The ATC murine model was genotyped by TPO-cre/ERT2; BrafCA/wt; Trp53ex2-10/ex2-10 and induced by intraperitoneal injection with tamoxifen. With the murine model, we investigated the tumor dynamics (tumor size ranged from 12.4 mm2 to 32.5 mm2 after 4 months of induction), survival (the median survival period was 130 days), and metastasis (lung metastases occurred in 91.6% of mice) curves and pathological features (characterized by Cd8, Foxp3, F4/80, Cd206, Ki67, and Caspase-3 immunohistochemical staining). The results indicated that spontaneous mATC possesses highly similar tumor dynamics and immunological microenvironment to human ATC tumors. In conclusion, with high similarity in pathophysiological features and unified genotypes, the mATC model resolved the shortage of clinical ATC tissue and sample heterogeneity to some extent. Therefore, it would facilitate the mechanism and translational studies of ATC and provide an approach to investigate the treatment potential of small molecular drugs and immunotherapy agents for ATC.

Protective effects of royal jelly and Echinacea against moxifloxacin-induced renal and hepatic injury in rats

Antibiotic use, especially fluoroquinolones, has been linked to extensive renal and hepatic injury thus inflicts a considerable health problem. Fifty rats were allocated into five groups (n = 10). Group 1 represented the normal-control group. Group 2 received moxifloxacin only (MOX; 8 mg/kg/day, i.p.) for seven days and represented the MOX-control group. Groups 3, 4, and 5 received MOX for seven days accompanied by royal jelly (RJ; 100 mg/kg/day, p.o.), Echinacea (ECH; 40 mg/kg/day, p.o.), and a combination of both at the aforementioned doses respectively for 30 days. All groups were investigated for renal and hepatic function tests. Renal tissue content of kidney injury molecule-1 (KIM-1) along with renal and hepatic tissue contents of reduced glutathione (GSH) and malondialdehyde (MDA) were assessed for all groups. Histopathological examination was performed followed by immunohistochemical staining for caspase-3 in renal and hepatic tissues. MOX administration resulted in significant renal and hepatic damage. RJ and ECH significantly improved the serum parameters of renal and hepatic functions along with increasing GSH and decreasing MDA in renal and hepatic tissues. Renal contents of KIM-1 were also reduced. Moreover, RJ, ECH, and their combination amended MOX-induced histopathological changes and significantly reduced caspase-3 immunohistochemical staining in both renal and hepatic tissues. The current study is the first to elucidate the effect of RJ, ECH, and their combination against MOX-induced renal and hepatic injury in rats. The study suggests that these protective effects are mainly via the reduction of oxidative stress induced by MOX administration.

Targeting Hydrogen Sulfide Modulates Dexamethasone-Induced Muscle Atrophy and Microvascular Rarefaction, through Inhibition of NOX4 and Induction of MGF, M2 Macrophages and Endothelial Progenitors.

Long-term use of Glucocorticoids produces skeletal muscle atrophy and microvascular rarefaction. Hydrogen sulfide (H2S) has a potential role in skeletal muscle regeneration. However, the mechanisms still need to be elucidated. This is the first study to explore the effect of Sodium hydrosulfide (NaHS) H2S donor, against Dexamethasone (Dex)-induced soleus muscle atrophy and microvascular rarefaction and on muscle endothelial progenitors and M2 macrophages. Rats received either; saline, Dex (0.6 mg/Kg/day), Dex + NaHS (5 mg/Kg/day), or Dex + Aminooxyacetic acid (AOAA), a blocker of H2S (10 mg/Kg/day) for two weeks. The soleus muscle was examined for contractile properties. mRNA expression for Myostatin, Mechano-growth factor (MGF) and NADPH oxidase (NOX4), HE staining, and immunohistochemical staining for caspase-3, CD34 (Endothelial progenitor marker), vascular endothelial growth factor (VEGF), CD31 (endothelial marker), and CD163 (M2 macrophage marker) was performed. NaHS could improve the contractile properties and decrease oxidative stress, muscle atrophy, and the expression of NOX4, caspase-3, Myostatin, VEGF, and CD31 and could increase the capillary density and expression of MGF with a significant increase in expression of CD34 and CD163 as compared to Dex group. However, AOAA worsened the studied parameters. Therefore, H2S can be a promising target to attenuate muscle atrophy and microvascular rarefaction.

Saffron extract attenuates Sofosbuvir-induced retinal neurodegeneration in albino rat.

Sofosbuvir is a novel drug candidate for the treatment of hepatitis C viral infection; however, vision loss is one of its growing adverse effects. Saffron is a natural biomolecule with a high antioxidant potential that has been efficiently used in some diseases caused by oxidative stress. This study evaluated Sofosbuvir's neurodegenerative effect on the retina of albino rat and examined the potential protective role of saffron aqueous extract. Twenty-one adult male albino rats were randomly divided into three groups: Control, Sofosbuvir-treated (41.1 mg/kg /day for 6 weeks), and Sofosbuvir + Saffron co-treated groups. Retinal specimens were biochemically analyzed for malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) levels. In addition, light and transmission electron microscopic examination, as well as immunohistochemical staining for Caspase-3, COX-2, and GFAP were performed. Sofosbuvir treatment caused a significant increase in retinal MDA, IL-6, and TNF-α levels coupling with a significant decrease in retinal total antioxidant capacity level. Histopathological findings revealed disturbed retinal architecture, detached pigment epithelium, vacuolated photoreceptors, in addition to a significant decrease in the thicknesses of both outer and inner nuclear layers, and the number of ganglionic cells. Ultrastructural examination revealed extensive degenerative changes in all retinal layers. Caspase-3, COX-2, and GFAP immunohistochemical expressions were significantly increased. Meanwhile, concomitant treatment with Saffron significantly improved retinal redox status, inflammation, histological, and ultrastructural parameters. Saffron may protect the retina from the hazardous effects of Sofosbuvir. Saffron could be used as an adjuvant therapy to protect patients receiving Sofosbuvir from retinal damage.

Meprin β mediates Osteopontin‐Signaling in IR‐Induced Acute Kidney Injury

Ischemia/reperfusion (IR) causes acute kidney injury (AKI) and is associated with high morbidity and mortality rates. Meprins metalloproteases are expressed abundantly in the brush border membranes of proximal kidney tubules and implicated in the progression of AKI. However, the underlying mechanisms are not fully understood. Meprins are capable to proteolytically processing osteopontin (OPN), a highly phosphorylated multifunctional and matricellular glycophosphoprotein. OPN mediates cell signaling pathways involved in apoptosis and extracellular matrix metabolism. The objective of the present study was to determine the effect of meprin β expression on OPN and downstream mediators of the OPN‐signaling pathway in IR‐induced renal injury. Surgical procedures were used to induce unilateral IR in wild‐type (WT) and meprin β knockout (βKO) mice. The mice were sacrificed at 24h post‐IR. Blood samples and kidney tissues were obtained for analysis. Neutrophil gelatinase associated lipocalin (NGAL) was used for biochemical assessment of kidney injury. Real‐time PCR, immunohistochemical staining, and western blot analysis were used to evaluate the levels of OPN, caspase‐3, Bcl‐2, and NFκB. Statistical analysis utilized 2‐way ANOVA. Our data show that NGAL levels were increased significantly in the kidney of WT (P=0.0001) and βKO (P=0.01) mice at 24h post‐IR when compared to their control counterparts. OPN mRNA increased in both genotypes at 24h post‐IR. Western blot analysis detected a full length OPN protein band at 66 kDa, with significant increases (p=0.001) in protein levels in WT kidney at 24h post‐IR when compared to levels in control kidneys. This increase in OPN was not observed in meprin βKO mice subjected to IR. Additionally, we detected a cleaved OPN fragment at 32 kDa, for which there were no significant changes in either genotype at 24h IR post‐IR. Immunohistochemical staining showed the increase in the levels of OPN occurred in select WT kidney tubules and in renal corpuscles. To determine whether meprin β expression correlates with the increased OPN, we used immunofluorescence counterstaining with proximal tubule (PT) markers (meprin β for WT and villin for βKO). Our data show that the increase in OPN occurred in both proximal and distal kidney tubules. Interestingly, higher levels of OPN were detected in the lumen of PTs from WT but not βKO kidneys, suggesting that meprin β is partly responsible for enhanced release of OPN into filtrate and ultimately into urine as previously reported. OPN functions as anti‐apoptotic protein in different diseases, therefore we evaluated its impact on caspase‐3, Bcl‐2, and NFκB. The mRNA levels for caspase‐3, Bcl‐2 and NFκB decreased in WT but increased in βKO mice at 24h post‐IR. Western blot detected higher levels of caspase‐3 and Bcl‐2 only in WT kidneys. Immunohistochemical staining for caspase‐3, Bcl‐2 and NFκB also showed higher levels in tubules and renal corpuscle of WT kidneys when compared to βKO kidneys at 24h post‐IR. Taken together, our data show that meprin β regulates OPN and down‐stream mediators of OPN‐signaling pathway in IR‐induced AKI.

Tanhuo Formula Inhibits Astrocyte Activation and Apoptosis in Acute Ischemic Stroke.

Tanhuo formula (THF), a traditional Chinese medicinal formula, has been demonstrated to be effective in the clinical treatment of acute ischemic stroke (AIS). However, its active ingredients, potential targets, and molecular mechanisms remain unknown. Based on the validation of active ingredient concentrations, our study attempted to elucidate the possible mechanisms of THF based on network pharmacological analysis and experimental validation. Components of THF were screened using network pharmacological analysis, and a compound–target network and protein–protein interaction (PPI) network were constructed. In total, 42 bioactive compounds and 159 THF targets related to AIS were identified. The PPI network identified AKT1, TNF, IL6, IL1B, and CASP3 as key targets. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated that the inflammation and apoptotic pathways were enriched by multiple targets. The main components of THF were identified via high-performance liquid chromatography. Subsequently, a validation experiment was conducted, and the expressions of GFAP, C3, TNF-α, and IL-6 were detected via immunofluorescence staining, confirming the inflammatory response at 30 min and 3 days post injury. Immunohistochemical staining for caspase-3 and TUNEL was also performed to assess apoptosis at the same time points. These results indicate that THF can effectively decrease neural cell apoptosis through the caspase-3 pathway and restrain excessive abnormal activation of astrocytes and the release of TNF-α and IL-6, which might be accompanied by the recovery of motor function. Thus, THF may serve as a promising therapeutic strategy for AIS through multiple targets, components, and pathways.

L-Carnitine alleviates hepatic and renal mitochondrial-dependent apoptotic progression induced by letrozole in female rats through modulation of Nrf-2, Cyt c and CASP-3 signaling

Letrozole (LTZ) is a non-steroidal aromatase inhibitor that is commonly used in breast cancer therapy. It has several side effects that might lead to the drug's cessation and data of LTZ's potential adverse effects on the hepatorenal microenvironment was conflicting. In addition, searching for therapeutic interventions that could modulate its adverse effects will be very beneficial. So, this study aims to determine the impact of LTZ on the hepatorenal microenvironment in cyclic female rats with a proposed regulatory role of L-Carnitine (LC) supplementation giving molecular insights into its possible mechanism of action. LTZ (1 mg/kg using 0.5% carboxy methyl cellulose as a vehicle for 21 consecutive days orally) to assess its impact on hepatorenal microenvironment. After treatment with LC (100 mg/kg orally) for 14 days, hepatorenal redox state (lipid peroxides (MDA), reduced glutathione (GSH) and catalase enzyme (CAT)), as well as relative gene expression of nuclear factor erythroid 2-related factor 2 (Nrf-2), cytochrome-c (Cyt c) and caspase-3 (CASP-3) were evaluated. Histopathological examination and immunohistochemical staining of CASP-3 in both liver and kidney were done. LTZ altered hepatic and renal functions. Relative gene expression of hepatorenal Nrf-2, Cyt c and CASP-3 as well as redox state revealed significant deterioration. Also, the liver and kidney tissues showed several micromorphological changes and intense reaction to CASP-3 upon immunohistochemical staining. It can be concluded that LC alleviates LTZ induced hepatorenal oxidative stress (OS) and mitochondrial-dependent apoptotic progression through modulation of Nrf-2, Cyt c, and CASP-3 signaling in female rats.

Therapeutic Effects of Bee Bread on Obesity-Induced Testicular-Derived Oxidative Stress, Inflammation, and Apoptosis in High-Fat Diet Obese Rat Model.

Obesity is a debilitating disorder with a variety of problems including oxidative stress, inflammation, and apoptosis. The aim of our study was to investigate the therapeutic role of bee bread on oxidative stress, apoptosis, and inflammation in the testis of obese rats. Thirty-two adult male Sprague Dawley rats, with weights between 230–300 g, were distributed into four groups (n = 8/group), namely normal control (C), obese (Ob), obese + BB or obese + OR [high-fat diet (HFD) for 6 weeks then HFD plus bee bread or orlistat for another 6 weeks] groups. Bee bread (0.5 g/kg) or orlistat (10 mg/kg/day) was diluted with distilled water and administered daily for 6 weeks by oral gavage. There were significant decreases in the activities of antioxidant enzymes [glutathione-S-transferase (GST), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione reductase (GR)], glutathione (GSH)] and total antioxidant capacity (TAC) levels and mRNA expressions of nuclear factor erythroid 2–related factor 2 (Nrf2), superoxide dismutase (Sod), catalase (Cat) and glutathione peroxidase (Gpx) in the obese group relative to the control group. Meanwhile, the mRNA levels of pro-inflammatory markers, namely: inducible nitric oxide synthase (Inos), nuclear factor kappa B (Nf-κβ), tumour necrotic factor α (Tnf-α) and interleukin 1β (Il-1β) were significantly increased while interleukin (Il-10) was decreased in the obese group relative to the control group. Further, proliferating cell nuclear antigen (PCNA) immunoexpressions decreased while cleaved caspase-3 immunohistochemical staining increased significantly in the obese group, in addition to increases in the mRNA levels of p53, Bax, Caspases-8, 9 and 3, relative to the control group. Treatment with bee bread showed increases in antioxidant enzymes and PCNA immunoexpression, as well as decreases in inflammation and apoptosis markers in the testes. This study has shown that bee bread has therapeutic effects against oxidative stress, inflammation, apoptosis in the testis of HFD-induced obese male rats, thereby suggesting its role as a natural supplement capable of treating obesity-induced male reproductive impairment.

Inonotus obliquus Aqueous Extract Suppresses Carbon Tetrachloride-induced Hepatic Injury through Modulation of Antioxidant Enzyme System and Anti-inflammatory Mechanism

Background: Chaga mushroom (Inonotus obliquus) is an edible macrofungus used in traditional and folk medicine for the treatment of various gastrointestinal disorders. It has shown potent anti-inflammatory, antioxidant and anticancer effects in several experimental studies, including anti-inflammatory and anticancer effects in colorectal cancer and intestinal inflammation. Whole extract or purified compound ergosterol peroxide from chaga mushroom showed anti-inflammatory mechanism via suppression of NF-κB/iNOS-COX-2 and growth inhibitory mechanism via regulation of apoptosis activation and β-catenin suppression. The diverse inflammatory and carcinogenic agents, like carbon tetrachloride (CCl4), a potent hepatotoxic chemical, cause liver damage by inducing lipid peroxidation and other oxidative damages. Aims: The study aimed to analyze the biochemical, cellular and molecular mechanisms of CCl4 induced chronic liver inflammation and carcinoma, and to analyze the effect of the extract of chaga mushroom on liver inflammation and cancer by virtue of anti-inflammatory mechanisms. Method: Physiological, histological and immunohistochemical analyses of the physiological functions and cellular functions were performed. Biochemical assays were conducted for assessing enzymatic changes in tissues. Molecular simulation and docking studies were performed for the evaluation of the molecular interaction. Results: CCl4-exposed mice exhibited a significant decrease in body weight followed by altered histopathological signatures in the liver. Supplementation of IOAE showed that treatment restored the normal structure of the tissues with large round nuclei in most of the cells. CCl4 caused a steep elevation in the levels of SGOT and SGPT to 2.32- and 1.8-fold as compared to control. The LDH level increased to 447 IU/L in CCl4 treated mice as compared to control (236 IU/L). Analysis of the oxidant enzyme pathway showed that CCl4 reduced the GSH level to 16.5 μM as compared to control (52 μM), and induced the catalase enzyme activity up to 259 U/mL as compared to control (124 U/L). These physiological and biochemical alterations were restored towards normal levels by IOAE administration. Immunohistochemical staining for caspase-3 and p53 showed that CCl4 notably increased their expressions, which were subsequently suppressed by administration of IOAE. The molecular simulation and docking studies using ergosterol peroxide from chaga mushroom with iNOS, COX-2 and TNF-α showed binding energy of -10.5, -8.9 and -9.1 Kcal/mol, respectively. These proteins interacting with ergosterol peroxide exerted an inhibitory effect on these critical proinflammatory signaling proteins. Conclusions: The results point out that IOAE is able to prevent damage of hepatic cells caused by CCl4 in mouse models through anti-inflammatory and growth inhibitory mechanisms, which can be utilized for natural prevention of liver toxicity.

Renovation of Intestinal Barrier by Polydatin in Experimentally Induced Ulcerative Colitis: Comparative Ultrastructural Study with L-Carnosine

Ulcerative colitis (UC) is a chronic inflammatory bowel disease associated with intestinal epithelial barrier impairment. Polydatin (PD), a natural product isolated from Polygonum cuspidatum, is known to have an anti-inflammatory, antioxidant, and antiapoptotic effect. We attempted to compare the protective impact of PD pretreatment on alterations to the intestinal epithelial barrier and the colonic wall’s ultrastructure accompanying ulcerative colitis to other conventional drugs in practice, primarily L-carnosine, which has not been addressed before. The rats were divided into 5 groups; 3 of them were treated with sulphasalazine (500 mg/kg), L-carnosine (30 mg/kg), and PD (45 mg/kg). All groups were administered their respective drugs 3 days before the UC was induced by acetic acid intra-rectally, and the treatment was continued until the 11th day. The disease activity index (DAI) was estimated, and a macroscopic scoring was established for the harvested colonic tissue. The tissues were extracted and processed for hematoxylin and eosin staining, caspase-3 immunohistochemical staining, electron microscopy, and biochemical analysis evaluating proinflammatory markers (IL-1β, TNF-α, and IL-6), myeloperoxidase (MPO), oxidative stress, and lipid peroxidation. Histopathological examination of colonic tissue showed that PD pretreatment effectively restored mucosal epithelial cells, intercellular tight junctions, goblet cells, and maintained the intestinal epithelial and endothelial barriers. PD suppressed MPO, proinflammatory markers, and malondialdehyde but enhanced superoxide dismutase and glutathione levels. It also hampered apoptosis, as evidenced by a reduction in caspase-3 expression. PD showed a significantly better response in preserving the intestinal epithelial barrier against acetic acid-induced colitis as compared to sulphasalazine and L-carnosine. These findings demonstrate the therapeutic role of PD for patients with UC.

Modulation of vigabatrin induced cerebellar injury: the role of caspase-3 and RIPK1/RIPK3-regulated cell death pathways

Vigabatrin is the drug of choice in resistant epilepsy and infantile spasms. Ataxia, tremors, and abnormal gait have been frequently reported following its use indicating cerebellar involvement. This study aimed, for the first time, to investigate the involvement of necroptosis and apoptosis in the VG-induced cerebellar cell loss and the possible protective role of combined omega-3 and vitamin B12 supplementation. Fifty Sprague-Dawley adult male rats (160-200g) were divided into equal five groups: the control group received normal saline, VG200 and VG400 groups received VG (200mg or 400mg/kg, respectively), VG200 + OB and VG400 + OB groups received combined VG (200mg or 400mg/kg, respectively), vitamin B12 (1mg/kg), and omega-3 (1g/kg). All medications were given daily by gavage for four weeks. Histopathological changes were examined in H&E and luxol fast blue (LFB) stained sections. Immunohistochemical staining for caspase-3 and receptor-interacting serine/threonine-protein kinase-1 (RIPK1) as well as quantitative real-time polymerase chain reaction (qRT-PCR) for myelin basic protein (MBP), caspase-3, and receptor-interacting serine/threonine-protein kinase-3 (RIPK3) genes were performed. VG caused a decrease in the granular layer thickness and Purkinje cell number, vacuolations, demyelination, suppression of MBP gene expression, and induction of caspases-3, RIPK1, and RIPK3 in a dose-related manner. Combined supplementation with B12 and omega-3 improved the cerebellar histology, increased MBP, and decreased apoptotic and necroptotic markers. In conclusion, VG-induced neuronal cell loss is dose-dependent and related to both apoptosis and necroptosis. This could either be ameliorated (in low-dose VG) or reduced (in high-dose VG) by combined supplementation with B12 and omega-3.

Meprin β Regulates Osteopontin‐Signaling in IR‐Induced Acute Kidney Injury

Ischemia/reperfusion (IR) is a leading cause of acute kidney injury (AKI) and associated with high morbidity and mortality rate. The underlying mechanisms involved in IR-induced AKI are not fully understood. Meprins, zinc metalloproteases that are abundantly expressed in the brush border membranes of proximal kidney tubules, have been implicated in the pathology of IR. Meprins are capable to proteolytically process several mediators of cell signaling pathways mediators involved in apoptosis and extracellular matrix (ECM) metabolism such as osteopontin (OPN). The objective of the current study was to determine how meprin β expression affects OPN and downstream mediators of the OPN-signaling pathway. Surgical procedures were used to induce unilateral IR with contralateral nephrectomy in wild-type (WT) and meprin β knockout (βKO) mice which are deficient in the meprin B (β-β) and the heteromeric isoform of meprin A (α-β). The mice were sacrificed at 24 h post-IR. Blood samples and kidney tissues were obtained for analysis. Neutrophil gelatinase associated lipocalin (NGAL) was used for biochemical assessment of kidney injury. Real-time PCR, immunohistochemical staining, and western blot analysis were used to evaluate the levels of OPN, caspase-3, Bcl-2, and NFκB. Statistical analysis utilized 2-way ANOVA. Our data show NGAL level were elevated significantly in WT (P=0.0001) and βKO (P=0.01) at 24 h post-IR compare to their control counterparts. However, the fold change was higher in WT when compared to βKO mice indicating that meprin β enhances kidney injury. OPN mRNA increased in both genotypes at 24 h post-IR. In contrast, OPN protein levels only increased in WT kidneys. Immunohistochemical staining showed the increase in the levels of OPN occurred in select WT kidney tubules. To determine whether meprins expression correlates with the increased OPN, we used immunofluorescence counterstaining with proximal tubule (PT) markers (meprin β for WT and villin for βKO). Our data show that the increase in OPN occurred in distal tubules (DTs), which do not express meprins. Interestingly, higher levels of OPN were detected in the lumen of PTs from WT but not βKO kidneys, suggesting that meprin β is responsible for enhanced release of OPN into filtrate and ultimately into urine as previously reported. OPN functions as anti-apoptotic molecules in different diseases, therefore we evaluated its impact on caspase-3, Bcl-2, and NFκB. The mRNA levels for caspase-3, Bcl-2 and NFκB decreased in WT but increased in βKO mice at 24 h post-IR. Immunohistochemical staining for caspase-3, Bcl-2 and NFκB also showed high staining levels in tubules of WT kidney compared to βKO at 24 h post-IR. These findings suggest that meprin β expression has a direct impact on the levels of OPN in the kidney and affects apoptotic genes regulated by the OPN signaling pathway.

Curcumin attenuates spatial memory impairment by anti-oxidative, anti-apoptosis, and anti-inflammatory mechanism against methamphetamine neurotoxicity in male Wistar rats: Histological and biochemical changes

ObjectiveMethamphetamine is used extensively around the world as a psychostimulant. The complications related to methamphetamine include methamphetamine-induced neurotoxicity, mainly involving intraneuronal processes, such as oxidative stress and excitotoxicity. Curcumin is effective against neuronal injury due to its antioxidant, anti-inflammatory effects. In this study, we examined the protective effects of curcumin against methamphetamine neurotoxicity. MethodsSixty male Wistar rats were divided into the following groups: control (n = 12), DMSO (n = 12), methamphetamine (n = 12), and methamphetamine + curcumin (100 and 200 mg/kg, respectively, intraperitoneal [IP]; n = 12). Neurotoxicity was induced by 40 mg/kg of methamphetamine administrated through 4 injections (4 × 10 mg/kg, q2h, IP). Curcumin (100 and 200 mg/kg) was administered at 7 days after the last methamphetamine injection. By using a Morris water maze task, the hippocampus-dependent memory and spatial learning were evaluated 1 day after the last curcumin injection. Then, the animal brains were isolated for biochemical measurements, as well as glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor protein-1(Iba-1) and caspase-3 immunohistochemical staining. ResultsThe current study demonstrated that administration of curcumin significantly attenuates spatial memory impairment (P < 0.01) following methamphetamine neurotoxicity. Curcumin caused a significant increase in the levels of superoxide dismutase and glutathione peroxidase (P < 0.05). However, it decreased tumor necrosis factor (TNF-α) (P < 0.05) and malondialdehyde (P < 0.01) levels as compared to the methamphetamine group. Also, curcumin significantly reduced Iba-1 (P < 0. 01), GFAP and caspase-3 positive cells in the hippocampus (P < 0.001). ConclusionCurcumin exerted neuroprotective effects on methamphetamine neurotoxicity because of its antioxidant and anti-inflammatory effect.

Morphologic assessment of hypertonic citrate adenine, histidine-tryptophan-ketoglutarate, and university of Wisconsin solutions for hypothermic uterus preservation in rats.

Optimizing perfusate for static cold storage is one of the key ways of reducing organ dysfunction and rejection in organ transplantation. Here, we tested the effectiveness of the three different solutions for hypothermic uterus preservation. Twenty rats were divided into four groups, five in each group. Uterine grafts were retrieved and perfused in situ. The uteri were preserved at 4°C in normal saline as control group (group NS), hypertonic citrate adenine (group HCA), histidine-tryptophan-ketoglutarate (group HTK), or university of Wisconsin solutions (group UW) for 0, 12, 24, and 48 h, respectively. HE, electron microscopy, TUNEL staining, and Cleaved Caspase3 immunohistochemical staining were assessed at each time point. There was no significant difference in the uterine retrieval time, perfusion time, and the amount of perfusion solution in NS, HCA, HTK, and UW groups (p > 0.05). HCA and HTK can well preserve the pathological morphology of rat uterine tissues for up to 24 h, and the apoptosis rates of the two groups are 7.2% and 7.1%, respectively, with no statistical difference (p > 0.05). Still, the protective effect of HTK on the ultrastructure of cells was much better than HCA. There was a significant difference in the apoptosis rate of UW (6.5%), HTK (8.8%), and HCA (9.4%) at 48 h, with mitochondrial and endoplasmic reticulum structure well preserved only in UW. At 4°C, normal saline is not suitable to preserve rat uterus for more than 12 h. The morphologic results would favor the use of HTK rather than HCA for short-term hypothermic uterus preservation (≤24 h). UW is better than HTK and HCA for 48 h hypothermic uterus preservation.

Phytochemical profile and protective effect of Ocimum basilicum aqueous extract in doxorubicin/irradiation-induced testicular injury.

Combined chemotherapy and radiotherapy usually associated with various comorbidities especially on rapidly proliferating cells as testis. This study aimed to characterize main constituents of Ocimum basilicum L. (OB) aqueous extract and examine its protective effect on doxorubicin/irradiation (DOXO/IR)-induced testicular injury in rats. Spectrophotometric analysis showed considerable amount of polyphenolic (146.31µg/mg) and flavonoid contents (28.63µg/mg); UPLC-ESI-MS/MS analysis revealed that the major flavonoid was apigenin-O-glucoside (7.53%) followed by luteolin (5.94%), while rosmarinic acid was the major polyphenolic (15.76%) followed by caftaric acid (9.39%); rutin and querctin were also present and were quantified using high-performance liquid chromatography. Administration of OB extract (200mg/kg per day; p.o.) to DOXO/IR rats resulted in marked improvement of associated testicular damage. Ocimum basilicum L. significantly decreased testicular levels of nuclear factor-kappa B and B-cell lymphoma-2 (Bcl2)-associated protein X, along with caspase-3 immunohistochemical staining. In addition, OB elevated testicular total antioxidant capacity, nuclear erythroid-related factor-2, Bcl2 and testosterone contents and Ki-67 immunohistochemical staining. Such changes were also accompanied by restoration of testicular architecture. The study highlights the protective role of OB aqueous extract in hampering most of the harmful chemotherapy/radiotherapy-induced outcomes via its antioxidant, antiapoptotic and cell regeneration abilities. Such findings may offer an incentive in expanding its use during chemotherapy and radiotherapy.