Thymoquinone: An Effective Natural Compound for Kidney Protection.

Kidney disease is a common health problem worldwide. Acute or chronic injuries may interfere with kidney functions, eventually resulting in irreversible kidney damage. A number of recent studies have shown that the plant-derived natural products have an extensive potential for renal protection. Thymoquinone (TQ) is an essential compound derived from Nigella Sativa (NS), which is widely applied in the Middle East as a folk medicine. Previous experiments have demonstrated that TQ has a variety of potential pharmacological effects, including anti-oxidant, antibacterial, antitumor, immunomodulatory, and neuroprotective activities. In particular, the prominent renal protective efficacy of TQ has been demonstrated in both in vivo and in vitro experiments. TQ can prevent acute kidney injuries from various xenobiotics through anti-oxidation, anti-inflammatory, and anti-apoptosis effects. In addition, TQ exhibited significant pharmacological effects on renal cell carcinoma, renal fibrosis, and urinary calculi. The essential mechanisms involve scavenging ROS and increasing anti-oxidant activity, decreasing inflammatory mediators, inducing apoptosis, and inhibiting migration and invasion. The purpose of this review is to conclude the pharmacological effects and the potential mechanisms of TQ in renal protection, shedding new light on the exploration of medicinal phyto-protective agents targeting kidneys.

Regulatory mechanism of macrophage polarization based on Hippo pathway.

Macrophages are found to infiltrate and migrate in a large number of Tumor-associated macrophages (TMEs) and other macrophages in the microenvironment of tumors and related diseases, and undergo phenotypic changes in response to a variety of cytokines, mainly including the primary phenotype M2 and the anti-tumor phenotype M1. The Hippo signaling pathway affects the development of cancer and other diseases through various biological processes, such as inhibition of cell growth. In this review, we focus on immune cells within the microenvironment of tumors and other diseases, and the role of the Hippo pathway in tumors on macrophage polarization in the tumor microenvironment (TME) and other diseases.

Reversing stage III oral adenocarcinoma in a dog treated with anti-canine PD-1 therapeutic antibody: a case report.

Monoclonal antibody targeting programmed cell death-1 (PD-1) is one of the most promising treatment therapies for human cancers. Canine PD-1 antibodies used in clinical trials have also shown efficacy in treating canine cancers. An 11-year-old male intact border collie presented to us for evaluation of left cervical mass. Computed tomography (CT) examination revealed an irregular pharyngeal mass invading the surrounding soft tissue. Histological and immunohistochemical results were consistent with a diagnosis of adenocarcinoma, most likely originating from the minor salivary gland. An anti-canine PD-1 monoclonal antibody was administered. Two months after the initial treatment, the tumor reached partial remission and maintained as such for 6 months. Finally, the patient was euthanized due to reasons unrelated to cancer, with a survival time of 316 days. To our knowledge, this is the first report of response to PD-1 blockade treatment in canine adenocarcinoma.

Prevalence, Risk Factors, Pathophysiology, Potential Biomarkers and Management of Feline Idiopathic Cystitis: An Update Review.

Feline idiopathic cystitis is a widespread disease in small animal clinics, which mainly presents with urinary signs like dysuria, stranguria, hematuria, pollakiuria, and periuria. The etiopathogenesis of the disease may involve interactions between the environmental stressors, neuroendocrine system and bladder of affected cats. Diagnostic biomarkers have not been tested in clinical studies though they are theoretically feasible, and since the clinical signs of the disease assemble those of other feline lower urinary diseases, its diagnosis is a procedure of exclusion. The primary treatment of the disease is long-term multimodal environmental modification (or enrichment) while anti-anxiety drugs and nutritional supplements are recommended for chronic recurrent cases. Still, many medicines need to be evaluated for their efficacy and safety. This review aims to provide readers with a comprehensive understanding of feline idiopathic cystitis by summarizing and updating studies concerning the prevalence, risk factors, etiological hypotheses, diagnostic procedures, possible treatments, and prognosis of the disease.

A Novel Canine Mammary Cancer Cell Line: Preliminary Identification and Utilization for Drug Screening Studies.

Canine malignant mammary tumor is a dangerously fatal neoplastic disease with poor survival in female dogs. The aim of this study was to preliminary characterize a novel canine mammary cancer cell line, B-CMT, from canine primary mammary gland tumor, and to utilize it as a cell model for in vitro screening of possible therapeutic drugs. The successfully established cell line, B-CMT, was cultured over 50 passages. B-CMT has a fast proliferation rate, and a population doubling time (PDT) of 33.6 h. The B-CMT cell line lacked human epidermal growth factor receptor-2 (HER-2), estrogen receptors (ER) and progesterone receptors (PR) expression by qRT-PCR. Compared with MDCK cells, CDH1 expression of CMT cell line was significantly decreased or even absent, but GATA3 expression dramatically increased, while TGF-ß expression was at a similar level. Interestingly, the B-CMT cell line from canine primary tumor also showed positive hypoxia inducible factor-1α (HIF-1α) results in immunofluorescence (IF), western blot, and qRT-PCR analysis. Ten days post inoculation with EGFP-B-CMT (B-CMT cells stably expressing EGFP), the experimental mice developed palpable soft tissue masses which histologically resembled the canine primary tumor, and was approved to be derived from B-CMT cell line through detection of EGFP by immunohistochemical (IHC) analysis. Moreover, we investigated the cytotoxicity of five drugs to B-CMT cells, and the results showed that rapamycin and imatinib significantly inhibited the proliferation of the cells in vitro within a certain range of concentration. They also induced cell cycle arrest of B-CMT cells at G1 and G2 phase, respectively. In summary, the results of this report showed that B-CMT cell line might serve as a tool for future studies on tumor microenvironment and drug resistance.

Protective effects of SP600125 on mice infected with H1N1 influenza A virus.

Influenza A virus (IAV) can cause high morbidity and mortality globally every year. Myriad host kinases and their related signaling pathways are involved in IAV infection, and the important role of the c-Jun N-terminal kinase signaling pathway during infection has been demonstrated. SP600125, an inhibitor of c-Jun N-terminal kinase, was found in our previous study to suppress IAV replication in vitro. In this study, we established a mouse model of H1N1 IAV infection and treated the mice with SP600125 to study its protective effect. The results showed that SP600125 treatment reduced the pulmonary inflammatory response, lung injury, and pulmonary viral load and increased the survival rate of H1N1-infected mice. Our data confirm the crucial role of c-Jun N terminal kinase in H1N1 virus replication and inflammatory responses in vivo. Hence, we speculate that SP600125 has a potential antiviral therapeutic benefit against IAV infection.

Characterization of porcine p53 and its regulation by porcine Mdm2.

Pigs have been increasingly recognized as a relevant model for studying many human diseases. However, functions and regulations of numerous critical molecules involved in human diseases are not well characterized in pigs, including the prominent tumor suppressor p53, a transcription factor involved in various anti-proliferative processes. In this study, we systematically characterized porcine p53 (p-p53) in its transcriptional activity and regulation by the E3 ligase Mdm2, in comparison with that of human p53 (h-p53). p-p53 is highly homologous to h-p53 with the N-terminal region showing relative divergence. p-p53 exhibits a comparable transcriptional activity to that of h-p53 towards a diverse range of known target genes, and is subject to ubiquitination and degradation by both human and porcine Mdm2 (h-/p-Mdm2). Utilization of the h-Mdm2 targeting compound Nutlin-3 and protein RPL11 inhibits the negative effect of p-Mdm2 on p-p53. These results suggest that the transcription activity and regulation of p-p53 is very similar to that of h-p53, and that the developed agents targeting the h-p53 pathway could be used in the study of p53 related processes and diseases in pigs.

Antimicrobial resistance of bacterial pathogens isolated from canine urinary tract infections.

Urinary tract infections (UTIs), many of which are caused by bacterial pathogens, are some of the most common infections in dogs. To effectively treat UTIs, it is important to identify the predominant bacterial pathogens and their susceptibility to antimicrobial agents. In this study, we collected 326 samples from cases with UTIs or other urinary system diseases at the China Agricultural University Veterinary Teaching Hospital, Beijing, from 2016-2018. In total, 129 non-duplicate bacterial isolates were recovered from 103 clinical samples. The proportion of positive female samples was higher than that of males. The predominant Gram-negative bacteria were Escherichia coli and Klebsiella spp., while Staphylococcus spp. were the predominant Gram-positive bacteria. Broth microdilution-based antimicrobial susceptibility testing showed that 39 % of E. coli and 51.5 % of Staphylococcus spp. isolates were multidrug-resistant. Specifically, E. coli isolates showed high rates of resistance to ampicillin (40.5 %), ceftazidime (59.5 %), and florfenicol (42.9 %), but limited resistance to amikacin (2.38 %), meropenem (7.14 %), and polymyxin E (7.14 %). In comparison, Staphylococcus spp. showed high rates of resistance to erythromycin (60.6 %), trimethoprim-sulfamethoxazole (54.6 %), and penicillin (45. 5 %), but low resistance rates to vancomycin (6.06 %) and nitrofurantoin (6.06 %). Pulsed-field gel electrophoresis (PFGE)-based typing identified 31 PFGE patterns among the 43 E. coli isolates. These results suggested that multiple bacterial strains, many of which are multidrug-resistant, can cause UTIs in dogs. Thus, basic antimicrobial susceptibility tests should be performed to provide guidance for the selection of first-line clinical therapeutics, and to help prevent the occurrence and spread of induced antimicrobial resistance.

HLA-B-associated transcript 3 (Bat3) stabilizes and activates p53 in a HAUSP-dependent manner.

The p53 pathway is a highly complex signaling network including several key regulators. HAUSP is a critical component of the p53 pathway acting as a deubiquitinase for both p53 and its key repressor Mdm2. Here, we identified a novel HAUSP-interacting protein, HLA-B-associated transcript 3 (Bat3) and found it to be capable of inducing p53 stabilization and activation via a HAUSP-dependent mechanism, resulting in cell growth inhibition. Surprisingly, the deubiquitylating enzymatic activity of HAUSP was not required for this phenomenon. Co-immunoprecipitation showed that p53 coexisted in a complex with Bat3 and HAUSP in vivo, and HAUSP may serve as a binding mediator to enhance the interaction between p53 and Bat3. Further studies revealed that formation of this three-protein complex interfered with the binding of p53 to its proteasome receptor S5a and promoted the accumulation of p53 in nucleus. Notably, Mdm2 protein abundance is also regulated by Bat3 in the presence of HAUSP. Overexpression of Bat3 and HAUSP increases Mdm2 protein levels without influencing the p53-Mdm2 interaction and Mdm2-mediated p53 ubiquitination, indicating that Bat3-HAUSP-mediated protein stabilization is not specific to p53 and different mechanisms may be involved in Bat3-mediated regulation of p53-Mdm2 pathway. Together, our study unravels a novel mechanism by which p53 is stabilized and activated by HAUSP-mediated interaction with Bat3 and implies that Bat3 might function as a tumor suppressor through the stabilization of p53.

Electroacupuncture improves microcirculation and neuronal morphology in the spinal cord of a rat model of intervertebral disc extrusion.

Most studies on spinal cord neuronal injury have focused on spinal cord tissue histology and the expression of nerve cell damage and repair-related genes. The importance of the microcirculation is often ignored in spinal cord injury and repair research. Therefore, in this study, we established a rat model of intervertebral disc extrusion by inserting a silica gel pad into the left ventral surface of T13. Electroacupuncture was used to stimulate the bilateral Zusanli point (ST36) and Neiting point (ST44) for 14 days. Compared with control animals, blood flow in the first lumbar vertebra (L1) was noticeably increased in rats given electroacupuncture. Microvessel density in the T13 segment of the spinal cord was increased significantly as well. The number of normal neurons was higher in the ventral horn of the spinal cord. In addition, vacuolation in the white matter was lessened. No obvious glial cell proliferation was visible. Furthermore, hindlimb motor function was improved significantly. Collectively, our results suggest that electroacupuncture can improve neuronal morphology and microcirculation, and promote the recovery of neurological functions in a rat model of intervertebral disc extrusion.

Short communication: molecular characterization of dog and cat p65 subunits of NF-kappaB.

Nuclear factor kappa B (NF-κB) plays an important role in the immune system. The p65 subunit is an important part of NF-κB unit, and studies of dog and cat p65 subunits of NF-κB (dp65 and cp65) are important in understanding their immune function. In this study, we described the molecular characterization of dp65 and cp65. The dp65 and cp65 complementary DNA encoded 542 and 555 amino acids, respectively, showing a high sequence homology with the mammalian p65 subunit (>87.5%). Quantitative polymerase chain reaction revealed that the p65 messenger RNA is highly expressed in the dog stomach and cat heart and adipose tissue. Functional NF-κB promoter-luciferase reporter vectors revealed that our isolated dp65 and cp65 cDNA encodes a functionally active protein. Transiently expressed dp65 and cp65 up-regulated pro-inflammatory cytokine expression levels in dog and cat, respectively. These findings suggest that dp65 and cp65 play important roles in regulating immune function.

Comparison of plasma lipoprotein profiles and malondialdehyde between hyperlipidemia dogs with/without treatment.

BACKGROUND: The aim of this study is to compare metabolic parameters, malondialdehyde as a lipid oxidation marker, and lipid profiles between dogs with untreated hyperlipidemia and hyperlipidemia with treatment, in order to examine the usefulness of malondialdehyde and lipid profiles as diagnostic parameters at early stages of hyperlipidemia. RESULTS: Dog samples were collected from four different veterinary clinics across Japan from March to June 2013. They were separated into three groups: control, untreated hyperlipidemia based on temporally screening, and hyperlipidemia with current anti-hyperlipidemic (statins and fibrates) treatment. Triglyceride levels of untreated hyperlipidemia dogs were significantly higher than those of control dogs. ALT levels of hyperlipidemic dogs with treatment were the highest among three groups. VLDL and LDL of both cholesterol and triglyceride of untreated hyperlipidemia dogs were the highest among three groups. HDL1 levels in triglyceride of hyperlipidemia dogs with treatment were significantly higher than those of control and untreated hyperlipidemia dog. Malondialdehyde concentrations of untreated hyperlipidemia dogs were significantly higher than those of control and hyperlipidemic dogs with treatment. CONCLUSIONS: In this study, dogs with untreated hyperlipidemia clearly showed abnormal lipid status, whereas hyperlipidemic dogs under anti-hyperlipidemia treatment showed more normal lipid status suggesting the effectiveness of the therapy. Anti-hyperlipidemics (statins and fibrates) for dogs are also effective in relieving elevated levels of lipids and lipid oxidation. Plasma lipid (triglyceride and cholesterol) profiles and malondialdehyde are useful diagnostic tools for identifying early stages of untreatment hyperlipidemia in dogs.

Change in mRNA expression of sirtuin 1 and sirtuin 3 in cats fed on high fat diet.

BACKGROUND: Mammalian sirtuins are homologs to the yeast silent information regulator 2 (Sir2), which is an NAD-dependent deacetylase. Sirtuins are comprised of 7 proteins, and each has different target proteins. Sirtuin 1 (SIRT1) plays important roles in maintaining metabolic functions and immune responses, and SIRT3 protects cells from oxidative stress-induced cell death. Both SIRT1 and SIRT3 are regulated by metabolic status and aging. Hence, SIRT1 and SIRT3 have been researched in metabolic diseases, such as type 2 diabetes mellitus (DM), fatty liver, and heart diseases. Although these diseases have been increasing, there is little information about relation between the diseases and SIRT1 and SIRT3 in cats. Therefore we cloned SIRT1 and SIRT3 cDNA, examined mRNA expression in cat tissues, and investigated the changes in SIRT1 and SIRT3 mRNA expression in peripheral blood leukocyte of cats fed on HFD for 6 weeks. RESULTS: Cat SIRT1 and SIRT3 contained a catalytic core region and showed high sequence homology with other vertebrate SIRT1 (>61.3%) and SIRT3 (>65.9%) amino acids. Real-time polymerase chain reaction analyses revealed that high expression levels were observed in the liver and skeletal muscle for SIRT1 and in the heart for SIRT3 in cats. In addition, both cat SIRT1 and SIRT3 expression levels in the pancreas were different between individuals. Cat SIRT1 mRNA expression in peripheral blood leukocytes was significantly elevated in obese cats fed on HFD (P < 0.05). CONCLUSIONS: Cat SIRT1 and SIRT3 genes are highly conserved among vertebrates, and HFD feeding may be related to SIRT1 mRNA expression mechanisms in cat peripheral blood leukocytes.

Long term intensive exercise training leads to a higher plasma malate/lactate dehydrogenase (M/L) ratio and increased level of lipid mobilization in horses.

Continuous high intensity training may induce alterations to enzyme activities related to glucose and lipid metabolism in horses. In our study, five Thoroughbred race horses (3 male and 2 female, avg age=5 yrs old) were compared against five riding horses (1 male, 1 female, 3 gelding, avg age=13 yrs old) in terms of energy metabolism, by examining plasma malate (MDH) and lactate (LDH) dehydrogenase activities and M/L ratio. MDH is involved in NADH and ATP generation, whereas LDH can convert NADH back into NAD(+) for ATP generation. An increase in plasma M/L ratio can reflect heightened energy metabolism in the liver and skeletal muscle of horses adapted to continuous intensive exercise. Moreover, plasma lipid metabolism analytes (adiponectin, NEFA, total cholesterol (T-Cho), and triglycerides (TG)) can reflect changes to lipolysis rate, which can also indicate a change in energy metabolism. Overall, race horses demonstrated increased MDH and LDH activity in plasma (4x and 2x greater, respectively), in addition to a plasma M/L ratio twice as high as that of riding horses (2.0 vs 1.0). In addition, race horses also demonstrated significantly higher levels of plasma NEFA (50% greater), TG (2x greater), and T-Cho (20% greater) as compared to riding horses. Therefore, race horse muscles may have adapted to prolonged high intensity endurance exercise by gaining a higher oxidative capacity and an increased capacity for fat utilization as an energy source, resulting in heightened energy metabolism and increased rate of lipid mobilization.

Supplementing five-point body condition score with body fat percentage increases the sensitivity for assessing overweight status of small to medium sized dogs.

BACKGROUND AND METHODS: Currently, five-point body condition scoring (BCS) is widely used by veterinarians and clinicians to assess adiposity in dogs in Japan. However, BCS score assignment is subjective in nature, and most clinicians do not score with half points, instead preferring to round off values, thereby rendering less accurate assessments. Therefore, we sought to determine whether assessing body fat percentage using simple morphometric measurements and supplementing this with five-point BCS can have increased sensitivity for detecting increasing adiposity in overweight small-medium sized dog breeds via plasma metabolite validation. RESULTS: Overall, lean body fat percentage was determined to be 15%-22% for male (non-neutered/neutered) dogs and 15%-25% for female (nonspayed/spayed). Dogs categorized as overweight by BCS had significantly higher levels of nonesterified fatty acids (P = 0.005), whereas animals categorized as overweight by BCS + body fat percentage were observed to have significantly higher levels of nonesterified fatty acids (P = 0.006), total cholesterol (P = 0.029), and triglycerides (P = 0.001) than lean animals. The increased sensitivity due to body fat percentage for gauging alterations in plasma metabolite levels may be due to increased correlation strength. Body fat percentage correlated positively with plasma insulin (r = 0.627, P = 0.002), nonesterified fatty acids (r = 0.674, P < 0.001), total cholesterol (r = 0.825, P < 0.0001), triglycerides (r = 0.5823, P < 0.005), blood urea nitrogen (r = 0.429, P < 0.05), creatinine (r = 0.490, P = 0.021), and total protein (r = 0.737, P < 0.0001) levels, which all tend to increase as a result of increasing adiposity. CONCLUSION: Supplementing body fat percentage with five-point BCS appears to increase the likelihood of validating overweight status in small-medium sized dog breeds by detecting changes in plasma metabolite levels, especially lipids, induced as a result of increasing adiposity.