Histopathological Observations Research Articles

Management of acute Aspergillosis in newly hatched chicks: A case report.

High and early mortalities of chicks often result in colossal economic losses. Aspergillosis, a fungal infection in chicks often occurs in the first 2 weeks of life and may be misdiagnosed as a common respiratory problem like Mycoplasmosis. Some Veterinarians are of the opinion that there is no effective way of treating Aspergillosis. This clinical case report describes the management of acute aspergillosis in chicks. Necropsies were done thrice. Grossly, congested livers, hemorrhagic nephritis, and congested lungs with whitish nodules were observed. Histologically, there were numerous multinuclear giant cells, macrophages and heterophils in the liver. The lung showed fungal hyphae and inflammatory cells. In the kidney, there was severe tubular epithelial necrosis indicated by the pyknotic nuclei, mononuclear infiltrates and haemorrhages. Considering the mortality rate, necropsy findings, microbial isolation and histopathologic observations, systemic Aspergillosis was diagnosed. The secondary bacterial infection was caused by Escherichia coli which was resistant to 8 antibiotics and susceptible only to Penstrep® (Penicillin +streptomycin). Initial resistance to copper sulphate necessitated a change to Nystatin which was used for 12 days via drinking water. Cumulative mortality was 757 (34.37%) in a flock of 2,202 chicks. Better control of systemic fungal infections may be enhanced by early application of anti-fungal susceptibility test.

White Spot Disease Outbreak in Confined Rain-fed Shrimp Culture Ponds of Chilka Lake: A Case Study

Incidence of large-scale mortality of cultured shrimp due to white spot disease caused by the white spot syndrome virus (WSSV) has been recorded from the confined rain-fed pond culture system in Haridas and Kusubenti villages of Chilka Lake, Orissa (India). Gross signs and symptoms of the disease and behavioural changes in shrimp were observed. Some of the soil and water quality parameters have been analysed and no adverse conditions could be observed. Detailed histopathological observations made on the moribund shrimp indicated the presence of WSSV. The probable source of the disease transmission in the confined culture system is discussed.

Effect of hexavalent chromium on growth performance and metabolism in broiler chicken.

Hexavalent chromium Cr (VI) is one of the most hazardous heavy metals in the environment and is toxic to living organisms causing tissue damage, disruption of the intestinal microbiota and cancer. However, there is little information on the relationship between the Cr (VI) and broiler chickens. The current study was performed to investigate the effect of Cr (VI) on growth performance, serum biochemical analysis, histopathological observations, and metabolomics analysis in broilers. Results show that Cr (VI) exposure significantly decreased the body weight (p < 0.01) and caused liver damages in broilers. With the extension of Cr (VI) action time, the liver appeared obvious pathological changes, including hepatic cord disorder, incomplete hepatocyte additionally, decreased serum biochemical indices of calcium (Ca), phosphorus (P), total protein (TP), phosphatase (ALP), and globin (GLB) significantly (p < 0.01). Moreover, metabolomics analysis indicated that 29 differential metabolites were identified, such as phytosphingosine, L-Serine, 12, 13-DHOME, Alpha-dimorphecolic acid, L-Methionine, L-Phenylalanine, 3-Dehydroshikimate, L-Tyrosine, and N-Acetyl-L-phenylalanine were significantly decreased under the action of Cr (VI) (p < 0.05). These 29 differential metabolites are mainly involved in 35 metabolic pathways, such as aminoacyl-tRNA biosynthesis, phenylalanine metabolism, sphingolipid, and linoleic metabolism. The study revealed that exposure to Cr (VI) resulted in a decrease in growth performance and metabolism, with the hazards and toxicity in broiler chicken. The findings provided new insight and a comprehensive understanding of the relationship between Cr (VI) and broiler chickens.

Role of Txnrd3 in NiCl2-induced kidney cell apoptosis in mice: Potential therapeutic effect of melatonin

Nickel (Ni) exposure is a significant risk factor for kidney dysfunction and oxidative stress injury in humans. Thioredoxin reductase 3 (Txnrd3), an important enzyme in animals, plays a role in maintaining cellular homeostasis and regulating oxidative stress. However, its protective effect against kidney injury has been determined. Melatonin (Mel) has antioxidant and anti-apoptotic effects and therefore may be a preventive and therapeutic agent for kidney injury. Our study aimed to investigate the roles of Mel and Txnrd3 in the treatment of nickel-induced renal injury. We divided 80 wild-type mice and 80 Txnrd3 -/- mice (C57BL/6 N) into a control group treated with saline, Ni group treated with 10 mg/kg NiCl2, Mel group treated with 2 mg/kg Mel, and Ni + Mel group given NiCl2 and Mel for 21 days. Histopathological and ultrastructural observation of the kidney showed that nuclei were wrinkled and mitochondrial cristae were broken in the Ni group, and these changes were significantly attenuated by Mel treatment. Mitochondrial and nuclear damage improved significantly in the Ni + Mel and Txnrd3-/- Ni + Mel groups. Furthermore, NiCl2 exposure decreased T-AOC, SOD, and GSH activities in the kidney. The decreases in antioxidant enzyme activity were attenuated by Mel, and these improvements were abolished by Txnrd3 knockout. NiCl2-induced increases in the mRNA and protein levels of apoptosis factors (Bax, Cyt-c, caspase-3, and caspase-9) were attenuated by Mel treatment, and Txnrd3 knockout abolished the repressive effect of Mel on apoptosis genes. Overall, we concluded that Mel improves oxidative stress and apoptosis induced by NiCl2 by regulating Txnrd3 expression in the kidney. Our results provide evidence for the role of Mel in NiCl2-induced kidney injury and identify Txnrd3 as a potential therapeutic target for renal injury.

Pectin encapsulated novel nanocomposite augments wound healing in Sprague Dawley rats

The complexity of the tissue remodeling process makes the treatment options for wound healing inadequate. Herein, the potential wound-healing activity of a novel nanocomposite with pectin and keratin incorporating silver nanoparticles and ferulic acid has been evaluated. Silver concentration, secondary structural stability of keratin, and the presence of ferulic acid and nano silver as a complex molecule within the nanocomposite were confirmed by XPS, CD spectroscopy, and ESI-MS analysis, respectively. The effects of the nanocomposite (NC) on cell migration and the role of apoptosis in wound healing were studied by scratch assay and (A.O./EtBr) staining, respectively. Subsequently, in vivo studies were carried out in male Sprague Dawley rats. Wound tracings measured exhibited significant wound contraction. Further, the relative gene expression of wound–healing biomarkers MMP2, MMP8, and KGF increased considerably in the 75 mg test group compared to the negative control group indicating a faster epithelialization phase of wound healing. Histopathological observations revealed remarkable skin regeneration in the test group. Altogether, the nanocomposite at 75 mg dose exhibits enhanced cell migration capability, reepithelialization and tissue remodeling activity than the standard, possibly due to the synergistic influence of the natural biomolecules. Therefore it is concluded that the NC exhibits accelerated wound healing with negligible toxicity.

Integrated network analysis and metabolomics reveal the molecular mechanism of Yinchen Sini decoction in CCl4-induced acute liver injury.

Objective: Yinchen Sini decoction (YCSND), a traditional Chinese medicine (TCM) formula, plays a crucial role in the treatment of liver disease. However, the bioactive constituents and pharmacological mechanisms of action remain unclear. The present study aimed to reveal the molecular mechanism of YCSND in the treatment of acute liver injury (ALI) using integrated network analysis and metabolomics. Methods: Ultra-high-performance liquid chromatography coupled with Q-Exactive focus mass spectrum (UHPLC-QE-MS) was utilized to identify metabolites in YCSND, and high-performance liquid chromatography (HPLC) was applied to evaluate the quality of four botanical drugs in YCSND. Cell damage and ALI models in mice were established using CCl4. 1H-NMR metabolomics approach, along with histopathological observation using hematoxylin and eosin (H&E), biochemical measurements, and reverse transcription quantitative real-time PCR (RT-qPCR), was applied to evaluate the effect of YCSND on CCl4- induced ALI. Network analysis was conducted to predict the potential targets of YCSND in ALI. Result: Our results showed that 89 metabolites in YCSND were identified using UHPLC-QE-MS. YCSND protected against ALI by reducing the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and malondialdehyde (MDA) contents and increasing those of superoxide dismutase (SOD), and glutathione (GSH) both in vivo and in vitro. The 1H-NMRmetabolic pattern revealed that YCSND reversed CCl4-induced metabolic abnormalities in the liver. Additionally, the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis identified five pathways related to liver injury, including the PI3K-AKT, MAPK, HIF-1, apoptosis, and TNF signaling pathways. Moreover, RT-qPCR showed YCSND regulated the inflammatory response (Tlr4, Il6, Tnfα, Nfκb1, Ptgs2, and Mmp9) and apoptosis (Bcl2, Caspase3, Bax, and Mapk3), and inhibited PI3K-AKT signaling pathway (Pi3k and Akt1). Combined network analysis and metabolomics showed a link between the key targets (Tlr4, Ptgs2, and Mmp9) and vital metabolites (choline, xanthine, lactate, and 3-hydroxybutyric acid) of YCSND in ALI. Conclusion: Overall, the results contribute to the understanding of the therapeutic effects of YCSND on ALI, and indicate that the integrated network analysis and metabolomics could be a powerful strategy to reveal the pharmacological effects of TCM.

Dietary supplementation of Patchouli oil increases the resistance against Streptococcus agalactiae infection in GIFT tilapia as revealed by transcriptome and 16s amplicon profiling

Large-scale disease outbreaks that caused by Streptococcus agalactiae (SA) infection have dramatically affected tilapia production worldwide. Patchouli oil (PO) is a traditional Chinese herbal medicine in treating gastrointestinal symptoms and is considered to be a safe and efficient candidate to treat SA infection in tilapia. In this study, we first investigated the changes of intestinal microbiota and host responses in GIFT tilapia after a 28-day dietary supplementation of different concentrations of PO (basic diet, 5 μL PO/g diet, or 15 μL PO/g diet) and followed by SA infection using an integrative analysis of transcriptome and 16 S amplicon profiling. We observed that dietary supplementation of 5 μL PO/g diet improved food intake and growth in tilapia. Histopathological observation confirmed there were increased surface area of intestinal villi and generally decreased ROS as revealed by the MAD, GSH-PX, and T-SOD activities in the tilapia intestines in response to dietary PO supplementation. The intestinal microbial diversities and community structures were significantly affected, and the colonization of probiotics (e.g., Cetobacterium and clostridium_ sensu_stricto_1) in the mucosa was improved in the dietary PO groups. The relative abundance of Streptococcus (0.033%) in the 15 μL PO/g dietary group was much lower than that in the control group (basic diet; 0.86%). A large number of DEGs were enriched in the immune-related pathways, e.g., lysosomal lumen, MHC protein complex, and toll-like receptor 2 signaling pathway. Our findings suggested that dietary supplementation of PO enhanced intestinal physical barriers, increased the colonization of probiotic bacteria and improved intestinal immunity in tilapia. This provides novel knowledge on PO as a dietary supplement to improve intestinal health against SA invasion in tilapia.

Metabolomics Analysis and Biochemical Profiling of Arsenic-Induced Metabolic Impairment and Disease Susceptibility.

Our study aimed to conduct a comprehensive biochemical profiling and metabolomics analysis to investigate the effects of arsenic-induced metabolic disorders, with a specific focus on disruptions in lipid metabolism, amino acid metabolism, and carbohydrate metabolism. Additionally, we sought to assess the therapeutic potential of resveratrol (RSV) as a remedy for arsenic-induced diabetes, using metformin (MF) as a standard drug for comparison. We measured the total arsenic content in mouse serum by employing inductively coupled plasma mass spectrometry (ICP-MS) after administering a 50-ppm solution of sodium arsenate (50 mg/L) in purified water. Our findings revealed a substantial increase in total arsenic content in the exposed group, with a mean value of 166.80 ± 8.52 ppb (p < 0.05). Furthermore, we investigated the impact of arsenic exposure on various biomarkers using enzyme-linked immunosorbent assay (ELISA) methods. Arsenic exposed mice exhibited significant hyperglycemia (p < 0.001) and elevated levels of homeostatic model assessment of insulin resistance (HOMA-IR), hemoglobin A1c (Hb1Ac), Inflammatory biomarkers as well as liver and kidney function biomarkers (p < 0.05). Additionally, the levels of crucial enzymes linked to carbohydrate metabolism, including α-glucosidase, hexokinase, and glucose-6-phosphatase (G6PS), and oxidative stress biomarkers, such as levels of glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), catalase, and superoxide dismutase (SOD), were significantly reduced in the arsenic-exposed group compared to the control group (p < 0.05). However, the level of MDA was significantly increased. Molecular analysis of gene expression indicated significant upregulation of key enzymes involved in lipid metabolism, such as carnitine palmitoyl-transferase-I (CPT-I), carnitine palmitoyl-transferase-II (CPT-II), lecithin-cholesterol acyltransferase (LCAT), and others. Additionally, alterations in gene expression related to glucose transporter-2 (GLUT-2), glucose-6-phosphatase (G6PC), and glucokinase (GK), associated with carbohydrate metabolism, were observed. Amino acid analysis revealed significant decreases in nine amino acids in arsenic-exposed mice. Metabolomics analysis identified disruptions in lipid metabolomes, amino acids, and arsenic metabolites, highlighting their involvement in essential metabolic pathways. Histopathological observations revealed significant changes in liver architecture, hepatocyte degeneration, and increased Kupffer cells in the livers of arsenic-exposed mice. In conclusion, these findings enhance our comprehension of the impact of environmental toxins on metabolic health and offer potential avenues for remedies against such disruptions.

Isolation, characterization and virulence of Metanophrys sp. (Ciliophora: Scuticociliatida) from large yellow croaker (Larimichthys crocea) in China

In recent years, an outbreak of large yellow croaker (Larimichthys crocea) disease caused by unknown ciliate infections has occurred along the southeast coast of China, resulting in substantial economic losses. To investigate the taxonomic status and pathogenic characteristics of the ciliate, we isolated the ciliate from the brain tissue of naturally diseased L. crocea larvae, established in vitro monoclonal culture, carried out molecular and morphological studies, and observed the host histopathology. In the present study, we characterized three linked genes (ITS1–5.8S-ITS2, LSU rDNA, SSU rDNA) of the ciliate. The mitochondrial cytochrome oxidase subunit I (mt-COI rDNA) and mitochondrial small subunit ribosome DNA (mt-SSU rDNA) were also applied. The phylogenetic analysis based on ITS1–5.8S-ITS2, LSU, SSU, mt-COI, and mt-SSU rDNA molecular markers showed that the ciliate had the closest genetic relationship with Metanophrys sinensis, and the sequence similarity was 89.95%, 98.22%, 98.74%, 94.63%, and 94.08%, respectively. It was also clustered in a branch with M. sinensis on the phylogenetic tree. Morphological observation showed that the ciliate was melon seed shaped, with a slightly sharp front end and a blunt rear end; a tail cilium, approximately 10 um long; and an oval macronucleus. There were 10–11 somatic kineties, and mitochondria were distributed beneath the cell surface membrane, forming a mitochondrial system that conforms to the morphological characteristics of Metanophrys. After artificially infecting the healthy L. crocea with the ciliate, a large number of ciliates with identical molecular markers and morphology were isolated from the infected L. crocea. The fins and skin of infected fish were partially ulcerated, and the tail or fin base was white, which is the same as the symptoms of the above-mentioned naturally diseased L. crocea. In addition, the results of histopathological observation showed that the ciliate could cause local necrosis and dissolution of L. crocea muscle fibers, infiltration of inflammatory cells in muscle spaces, increased swelling cells in gills and skin, and vacuolation of liver and spleen cells. Therefore, the ciliate that causes the above disease can be classified into the genus Metanophrys, which has been isolated and identified for the first time.

Histopathological Observations on Liver and Gills of Clarias gariepinus Juveniles Treated with Some Prophylactics

This study investigated histopathological changes on the liver and gills of C. gariepinus juveniles treated intermittently with different concentrations of prophylactics. Potassium permanganate (T2), Hydrogen peroxide (T3) and Formalin (T4) were used as prophylactics and administered to 15 fish each at concentrations of 2 ppm, 150 ppm, and 100 ppm respectively. The treatments which were in three replicates each with a control set up (T1) were repeated biweekly for 10 weeks. The histomacrographs of the liver and the gills show that there were no visible alterations on the structures of the tissues of fish in the control experiment (T1). However, major histopathological observations for the fish in T2, T3, and T4 were severe degeneration in the gill structure, fusion of the gill rakers, slight changes in the hepatocytes, diffuse vacuolation of hepatocytes. Among the used chemicals, potassium permanganate appeared to have the least negative effects on the tissues and it is therefore recommended at 2 ppm for prophylactic treatment of fish against diseases.

Exploration of the anti-diabetic potential of hydro-ethanolic leaf extract of Koenigia polystachya L.: an edible wild plant from Northeastern India

BackgroundGlobally, medicinal plants are used to treat diseases like diabetes. The present study evaluates the possible antioxidant, acute oral toxicity, the in-vitro and in-vivo antidiabetic potential of the hydro-ethanolic leaf extract of Koenigia polystachya (HELeKP) against beta-cell damage in experimentally induced diabetes mellitus. The DPPH (2,2-diphenyl-1-picrylhydrazine), ABTS [2,2′-azino bis-(3-ethylbenzothiazoline-6-sulfonic acid)], H2O2 (Hydrogen peroxide), superoxide radical scavenging activity and NO (Nitric oxide) assay estimated the in-vitro antioxidant assay of HELeKP. The acute oral toxicity study was evaluated per the OECD (Organization for Economic Cooperation and Development) test guidelines 425. Diabetes was stimulated in rats with a single dose of Streptozotocin (STZ), and after confirmation of diabetes, HELeKP was given orally for 21 days. Blood/serum samples were gathered and examined for biochemical changes, while tissue samples were evaluated for histopathological alterations.ResultsThe IC50 value of the HELeKP for all the anti-oxidant assays confirms the free radical scavenging activity. The data on acute oral toxicity revealed that the HELeKP used in the study was comparatively very safe. The outcomes of the in-vivo study suggested that the extract significantly reduced (p < 0.001) the fasting glucose level in STZ-induced diabetic rats. Furthermore, the lipid profile level was significantly normalized (p < 0.01, p < 0.001) in diabetic rats. The histopathological observation of the pancreas in HELeKP-treated rats showed significant beta-cell restoration.ConclusionsBased on the outcomes of this study, the HELeKP-treated rats have significant free radical scavenging and anti-diabetic potential. Therefore, it can be recommended as a beneficial functional vegetable for consumption.

Effect of Anthocyanin on the Ovarian Damage and Oxidative Stress Markers in Adult Female Rat After Torsion/Detorsion

Objectives: This experimental study aimed to explore the impact of anthocyanin on histopathological alterations and oxidative stress induced by ovarian torsion/detorsion in mature rats. Materials and Methods: A total of 32 female Wistar rats were categorized into four groups: G1 (sham), G2 (ovarian torsion endured for 3 hours followed by detorsion), G3 (ovarian torsion persisted for 3 hours with prior administration of 100 mg/kg anthocyanin followed by detorsion), and G4 (received 100 mg/kg anthocyanin). After a reperfusion interval of 10 days, blood specimens were obtained for quantifying estrogen, testosterone, and oxidative stress markers in the plasma. Histomorphometric analysis was done to evaluate post-procedural tissue alterations. Results: The histopathological observations revealed considerable degenerative modifications in the G2 group (subjected to torsion/ detorsion) compared to the other groups (P&lt;0.001). Estrogen, glutathione peroxidase (GPX), and superoxide dismutase (SOD) levels displayed notable reduction in the G2 group; however, these levels were reinstated in the anthocyanin-treated groups. The level of malondialdehyde (MDA), an indicator of oxidative stress, surged during the ischemic phase (P&lt;0.001). Conclusions: Administration of anthocyanin led to a decline in MDA levels across the other groups. These findings collectively suggest that anthocyanin supplementation holds the potential to shield ovarian tissue against oxidative stress and structural damage incurred as a consequence of torsion/detorsion events.

Non-O1/O139 Vibrio cholerae causes severe intestinal disease in bullfrogs (Rana catesbeiana)

Bullfrogs (Rana catesbeiana) are amphibians with high economic value, but in recent years, bullfrog farming has encountered serious threats of bacterial diseases, and the “bullfrog economy” is facing a continuous decline. In this study, the dominant strain was isolated from diseased bullfrogs in a bullfrog farm in Nanning, Guangxi, and based on its morphological, physiological, and biochemical characteristics and analysis of 16S rRNA gene sequences, the strain was identified as a non-O1/O139 group Vibrio cholerae and named TC1. Three virulence factors were identified in this strain, including hemolysin, outer membrane protein, and toxin-coregulated pili. Drug susceptibility testing showed that the strain resisted gentamicin, florfenicol, nitrofural, oxytetracycline, neomycin, penicillin, amoxicillin, doxycycline, and sulfamonomethoxine. The results of artificial infection experiments showed that TC1 caused serious pathologies such as abdominal swelling and anal prolapse in bullfrogs, especially severe intestinal bleeding. Histopathological observations revealed that the bullfrog intestine exhibited obvious pathological lesions. These results provide an essential epidemiological basis for controlling V. cholerae infections in aquatic animals and demonstrate the promise of bullfrogs as an amphibian model for studying the pathogenesis of V. cholerae.

Network analysis and experimental approach to investigate the potential therapeutic mechanism of zishen yutai pills on premature ovarian insufficiency

BackgroundAs society continues to develop, women are more at risk of gonadotoxic substance exposure. Consequently, the incidence of premature ovarian insufficiency (POI) has increased significantly in the past decades. Hormone replacement therapy (HRT) is recommended as the standard treatment to relieve hypoestrogenic symptoms; however, its potential side effects and contraindications have drawn widespread controversy and concern. As such, the Chinese medicine Zishen Yutai Pill (ZSYTP) commonly used for treating miscarriage and menoxenia, is a highly promising alternative drug candidate against POI, however its therapeutic mechanism has not been completely elucidated. ObjectiveTo systematically analyze the potential therapeutic targets of ZSYTP on POI, we combined network pharmacology analysis and molecular docking to predict critical target genes, with experimental validation on POI murine models. MethodsThe active compounds of ZSYTP were collected from three online databases, and the candidate targets were predicted based on the chemical structure. The POI-related targets were obtained from four databases. A PPI network was constructed to find the key target genes between ZSYTP and POI, while GO and KEGG enrichment analyses were employed to study the mechanism of ZSYTP against POI. The binding capability of the key co-targets with active components was examined by molecular docking. We used a cyclophosphamide (CTX)-inducible POI mouse model to verify our predictions by histopathological observation, immunohistochemical staining (caspase-3, TUNEL assay), hormone determination (FSH, AMH) and ribonucleic acid sequencing (RNA Seq). Progynova was also used to study the difference between ZSYTP and HRT. ResultWe identified 21 target genes as the hub between ZSYTP and POI. The GO and KEGG analyses revealed that the molecular mechanism of ZSYTP against POI were mainly based on the regulation of gene and protein expression. A variety of signaling pathways may be involved in the treatment of ZSYTP against POI, especially PI3K-AKT, HIF-1 and the AGE-RAGE cascades. Docking simulation showed that G1, C1, SR5, and F1 had relatively lower binding energy. In vivo, ZSYTP significantly reversed CTX-induced ovarian damage in follicle number, hormone level and apoptosis, with an overall improved therapeutic effect compared to Progynova. Results from RNA-Seq revealed that the PI3K-AKT, Hippo, AGE-RAGE, and Rap1 signaling pathways and regulation of inflammation, immune response, and lipid metabolism may mediate the protective effects of ZSYTP against POI, which is different than Progynova's mechanism of action. ConclusionsCollectively, this study indicates that ZSYTP could be a highly promising alternative as a non-HRT-based therapy for POI. Its mechanism involves multiple signaling pathways, alleviating ovarian apoptosis and recovering AMH and FSH level. However, the discrepancy between different research techniques highlight the necessity of further experimental verification from other aspects such as translation and posttranslational modification.

Croton hirtus attenuating streptozotocin-induced neuroinflammation in rats

ObjectiveThe present study was aimed to investigate the neuroprotective effect of Croton hirtus (CH) extract against streptozotocin (STZ) in rats. Methods(i) The sub-chronic toxicity consisted of 24 adult rats of either sex weighing from 160 to 200 g were divided into four groups with six rats in each group. Rats in group 1 received Dimethyl sulfoxide (DMSO) mixed with saline; rats in groups 2, 3, and 4 received 100, 200, and 400 mg/kg of methanolic extract of CH (MECH) orally by gavage administration for 28 d, respectively. The functional observation battery and locomotor activity were graded as part of their neurobehavioral activity and the brain regions, including cortex and hippocampus, were analyzed for neuropathological abnormalities. (ii) The main research consisted of 30 adult male Wistar rats weighing from 160 to 200 g, which were divided into five groups and six rats in each group, including control (I), STZ (II), Donepezil (III), MECH (100 mg/kg, IV), and MECH (200 mg/kg, V) groups. Rats in group I received citrate buffer orally and DMSO mixed with saline for 14 d. Rats in group II received STZ via intracerebroventricular injection (3 mg/kg, bilateral ICV-STZ) on days 1 and 3 followed by DMSO mixed with saline for 14 d. Rats in groups III, IV, and V received STZ administration on days 1 and 3 followed by Donepezil [3 mg/(kg·d), p.o.] and MECH [100 and 200 mg/(kg·d), p.o.] for 14 d. Rats were tested for learning and memory parameters such as Morris water maze (MWM) and passive avoidance test (PAT). They were sacrificed after completing behavioural experiments; brains were harvested to estimate the acetylcholinesterase (AChE), lipid peroxidation (LPO), glutathione (GSH), and superoxide dismutase (SOD) by using UV-Visible Spectrophotometer; caspase-3 was evaluated by total fluorescence emission spectra; amyloid β (Aβ) levels were detected using enzyme-linked immuosorbent assay (ELISA); and histopathological examination was conducted in the CA1 region of the hippocampus. Results(i) The sub-chronic toxicity results revealed that open field test parameters including line crossing, rearing, entering the middle square, defecating, or urinating did not differ significantly in the MECH rats (P > 0.05). The histopathological observation did not show any lesions in the neuronal cells and inflammation in both the regions in MECH rats compared with control rats. (ii) The main study findings demonstrated that STZ-treated rats showed a significant increase in impairment in learning and memory parameters (P < 0.001), the levels of AChE, caspase-3, Aβ (1-40 and 1-42), and LPO were increased significantly (P < 0.001), and significant decrease was found in the levels of SOD (P < 0.001) and GSH (P < 0.01). Moreover, neuronal damage was found in the hippocampus. In contrast, STZ-induced behavioural and biochemical impairments in rats were considerably decreased by treatment with MECH dose-dependently. ConclusionMECH significantly prevented the memory deficit induced by STZ due to antioxidant action. Restoration of cholinergic functioning may be the cause of behavioural improvement. Therefore, MECH may be able to treat cognitive disorders like Alzheimer's disease (AD).

Toxicological Evaluation and Pain Assessment of Four Minor Cannabinoids Following 14-Day Oral Administration in Rats.

Introduction: Despite growing consumer interest and market availability, the safety of minor cannabinoids, generally present in low concentrations in Cannabis sativa L., is not well understood. Materials and Methods: Cannabichromene (CBC; 3.2, 10, 17, 22, 32, or 100 mg/kg-bw/day), cannabinol (CBN; 1, 3.2, 10, 17, 32, or 100 mg/kg-bw/day), delta-8-tetrahydrocannabinol (D8-THC; 0.32, 1, 3.2, or 10 mg/kg-bw/day), tetrahydrocannabivarin (THCV; 3.2, 10, 17, 22, 32, or 100 mg/kg-bw/day), and vehicle (medium-chain triglyceride oil) preparations were administered via oral gavage once daily for 14 days to Sprague Dawley rats. Changes in behavior, body weight, food consumption, clinical pathology, organ weights, body temperature, and thermal pain sensitivity (tail flick assay) were assessed. Select organ tissues were collected at terminal necropsy and fixed for histopathological examination. Results: No treatment-related deaths were observed throughout the study, and cannabinoids were generally well tolerated. While some significant trends in body weight differences from controls (increases and decreases) were observed, these occurred independently of food consumption. Overall, differences in serum chemistry and hematology parameters between cannabinoid groups and their respective control groups were considered to occur due to biological variation among rats. No treatment-related gross abnormalities were observed in examined organs. Significant changes in absolute and relative organ weights occurred primarily in males and were generally of negligible magnitude. There were no biologically significant histopathological observations. While pain tolerance was significantly improved in animals treated with D8-THC (3.2 and 10 mg/kg-bw/day, day 14), results across minor cannabinoids were inconsistent and warrant further study. Conclusion: Minor cannabinoids were well tolerated across 14 days of daily oral administration at the doses assessed. Modest, dose-dependent trends in relative organ weights and serum chemistry parameters warrant exploration at higher oral doses. These data will assist in dose selection for future studies investigating the long-term safety and effects of CBC, CBN, D8-THC, and THCV.

Suppression of colon cancer growth by berberine mediated by the intestinal microbiota and the suppression of DNA methyltransferases (DNMTs).

The purpose of this study was to demonstrate the regulatory effect of berberine (BBR) on the intestinal microbiota and related epigenetics during the inhibition of colon cancer cell growth in vitro and in vivo. We used a nude mouse xenograft model with HT29 colon cancer cells to establish and divide into a model group and BBR group. The mice were treated for four weeks, and HT29 cells in the BBR group were cultured for 48h. Cetuximab and the DNA transmethylase (DNMT) inhibitor 5-AZA-dC were added to HT29 cells. Tumour volume and weight were measured by hematoxylin-eosin (HE) staining for histopathological observation. Mouse faeces were collected, and the gut microbiota was analysed with 16S rDNA amplicons. The levels of cytokines in the supernatant of HT29 cells were measured by ELISA. A CCK-8 kit was used to examine the proliferation of HT29 cells, and RT‒PCR was used to measure the levels of c-Myc, DNMT1, DNMT3A, and DNMT3B. We found that BBR reduced the growth of colon cancer cells to a certain extent in vitro and in vivo, although the difference was not statistically significant compared with that in the model group. BBR significantly mediated the abundance, composition and metabolic functions of the intestinal microbial flora in mice with colon cancer. The effect of BBR on inflammatory cytokines, including IL-6, FGF, and PDGF, was not obvious, but BBR significantly downregulated IL-10 levels (P < 0.05) and reduced c-Myc, DNMT1, and DNMT3B levels (P < 0.05). Inhibiting DNMTs with 5-AZA-dC significantly suppressed the proliferation of HT29 cells, which was consistent with the effect of BBR. The inhibitory effect of berberine on colon cancer is related not only to the intestinal microbiota and its metabolic functions but also to the regulation of DNMTs.

Apoptosis Induction by New Coumarin Derivatives in a Mice Model of Breast Cancer

In the last decades, numerous studies have focused on the search for new agents to suppress the growth of cancer cells. In this study, we investigated the effect of two novel synthetic coumarin derivatives, namely 2-amino-4-(4-(2-hydroxyethoxy)-3-methoxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile and 2-amino-4-(4-hydroxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]coumarin-3-carbonitrile, on the induction of apoptosis in breast cancer in a mouse model. Breast cancer was induced in BALB/c mice, which were randomly divided into six groups and then underwent the experiment. The groups and treatments included A1: coumarin A with a low dose (10 µm), A2: coumarin A with a high dose (1 mM), B1: coumarin B with a low dose (10 µm), B2: coumarin B with a high dose (1 mM), D: doxorubicin, and C: cancer control/ treatment with normal saline. The samples underwent treatments for 5 weeks. Animals were euthanized, and tissue samples, including the lung, liver, and tumor mass, were collected for histopathological examination. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine some apoptotic markers, such as BCL-2, caspase-9, COX-2, and c-Myc. The qRT-PCR presented that both coumarin compounds could significantly alter the expression levels of BCL-2, caspase-9, COX-2, and c-Myc. Consistent with these results, histopathological observations showed a significant reduction in pathological lesions and severity of malignancy of the tumor mass, as well as a decrease in microscopic metastases in the lung and liver. This suggests that the present new coumarin compounds may induce apoptosis in breast cancer cells by altering some apoptosis-related genes that may play a chemotherapeutic role in breast cancer therapy in the future.

68Ga-FAPI-04 Positron Emission Tomography Distinguishes Malignancy From 18F-FDG-Avid Colorectal Lesions

Lesions with a high uptake of 18F-fluorodeoxyglucose (18F-FDG) on positron emission tomography-computed tomography (PET-CT) can be benign and malignant. New radiotracers, such as the gallium 68 (68Ga)-labeled fibroblast activation protein inhibitor 4 (FAPI-04), could be used to diagnose colorectal carcinoma. This study aimed to evaluate the efficacy of 68Ga-FAPI-04 PET in differentiating benign from malignant 18F-FDG-avid colorectal lesions. An azoxymethane/dextran sodium sulfate (AOM/DSS)-induced rat colorectal tumor model was developed. Double-tracer 68Ga-FAPI-04 and 18F-FDG PET-CT were applied in the rat model and 22 patients. The PET-CT data were analyzed with enteroscopy, histopathologic observations, immunohistochemistry (IHC) staining, and radioautography results. One hundred seventy-two patients with pathologically confirmed colorectal lesions were enrolled in FAP IHC staining. We found that 68Ga-FAPI-04 PET-CT imaging accurately distinguished the malignant from benign inflammatory lesions in an AOM/DSS-induced rat colorectal tumor model. Of 22 patients with gastric cancer but without colorectal carcinoma, 8 had 18F-FDG uptake in the colorectum, but 68Ga-FAPI-04 PET was negative in these sites. An inflammatory lesion or adenoma did not interfere with 68Ga-FAPI-04 PET imaging. Among the 18F-FDG-avid colorectal lesions, 80 of 94 pathologically malignant lesions (85.1%) were FAP-positive, and only 16 of the 78 premalignant or benign lesions (20.5%) had a weak 68Ga-FAPI-04 uptake. 68Ga-FAPI-04 PET-CT could be used to distinguish between benign and malignant 18F-FDG-avid colorectal lesions.

Potential of phage EF-N13 as an alternative treatment strategy for mastitis infections caused by multidrug-resistant Enterococcus faecalis

Bovine mastitis is the most common and costly disease affecting dairy cattle throughout the world. Enterococcus faecalis is one of the environmental origin mastitis-causing pathogens. The treatment of bovine mastitis is primarily based on antibiotics. Due to the negative impact of developing antibiotic resistance and adverse effects on soil and water environments, the trend toward use of nonantibiotic treatments is increasing. Phages may represent a promising alternative treatment strategy. However, it is unknown whether phages have therapeutic effects on E. faecalis-induced mastitis. Thus, the objective of this study was to investigate the degree of protection conferred by a phage during murine mastitis caused by multidrug-resistant E. faecalis. Enterococcus faecalis was isolated from the milk of dairy cows with mastitis, and a phage was isolated using the E. faecalis isolates as hosts. The bactericidal ability of the phage against E. faecalis and the ability to prevent biofilm formation were determined in vitro. The therapeutic potential of the phage on murine mastitis was evaluated in vivo. We isolated 14 strains of E. faecalis from the milk of cows with mastitis, all of which exhibited multidrug resistance, and most (10/14) could form strong biofilms. Subsequently, a new phage (EF-N13) was isolated using the multidrug-resistant E. faecalis N13 (isolated from mastitic milk) as the host. The phage EF-N13 belongs to the family Myoviridae, which has short latent periods (5 min) and high bursts (284 pfu/cell). The genome of EF-N13 lacked bacterial virulence-, antibiotic resistance-, and lysogenesis-related genes. Furthermore, bacterial loading in the raw milk medium was significantly reduced by EF-N13 and was unaffected by potential IgG antibodies. In fact, EF-N13 could effectively prevent the formation of biofilm by multidrug-resistant E. faecalis. All of these characteristics suggest that EF-N13 has potential as mastitis therapy. In vivo, 1 × 105 cfu/gland of multidrug-resistant E. faecalis N13 resulted in mastitis development within 24 h. A single dose of phage EF-N13 (1 × 104, 1 × 105, or 1 × 106 pfu/gland) could significantly decrease bacterial counts in the mammary gland at 24 h postinfection. Histopathological observations demonstrated that treatment with phage EF-N13 effectively alleviated mammary gland inflammation and damage. This effect was confirmed by the lower levels of proinflammatory cytokines IL-6, IL-1β, and tumor necrosis factor-α in the mammary gland treated with phage EF-N13 compared with those treated with phosphate-buffered saline. Overall, the data underscored the potential of phage EF-N13 as an alternative therapy for bovine mastitis caused by multidrug-resistant E. faecalis.