Antidiabetic and Renoprotective Effects of Coffea arabica Pulp Aqueous Extract through Preserving Organic Cation Transport System Mediated Oxidative Stress Pathway in Experimental Type 2 Diabetic Rats.

Coffea arabica pulp (CP) is a by-product of coffee processing. CP contains polyphenols that have exhibited beneficial effects, including antioxidant and lipid-lowering effects, as well as enhanced insulin sensitivity, in in vitro and in vivo models. How polyphenols, as found in CP aqueous extract (CPE), affect type 2 diabetes (T2D) has not been investigated. Thus, the present study examined the potential antidiabetic, antioxidant, and renoprotective effects of CPE-rich polyphenols, using an experimental model of T2D in rats induced by a high-fat diet and a single low dose of streptozotocin. The T2D rats received either 1000 mg/kg body weight (BW) of CPE, 30 mg/kg BW of metformin (Met), or a combination treatment (CPE + Met) for 3 months. Plasma parameters, kidney morphology and function, and renal organic transport were determined. Significant hyperglycemia, hypertriglyceridemia, insulin resistance, increased renal lipid content and lipid peroxidation, and morphological kidney changes related to T2D were restored by both CPE and CPE + Met treatments. Additionally, the renal uptake of organic cation, 3H-1-methyl-4-phenylpyridinium (MPP+), was reduced in T2D, while transport was restored by CPE and CPE + Met, through an up-regulation of antioxidant genes and protein kinase Cα deactivation. Thus, CPE has antidiabetic and antioxidant effects that potentially ameliorate kidney function in T2D by preserving renal organic cation transport through an oxidative stress pathway.

Tiliacora triandra (Colebr.) Diels Leaf Aqueous Extract Inhibits Hepatic Glucose Production in HepG2 Cells and Type 2 Diabetic Rats.

This study investigated the effects of Tiliacora triandra (Colebr.) Diels aqueous extract (TTE) on hepatic glucose production in hepatocellular carcinoma (HepG2) cells and type 2 diabetic (T2DM) conditions. HepG2 cells were pretreated with TTE and its major constituents found in TTE, epicatechin (EC) and quercetin (QC). The hepatic glucose production was determined. The in vitro data were confirmed in T2DM rats, which were supplemented daily with 1000 mg/kg body weight (BW) TTE, 30 mg/kg BW metformin or TTE combined with metformin for 12 weeks. Results demonstrate that TTE induced copper-zinc superoxide dismutase, glutathione peroxidase and catalase genes, similarly to EC and QC. TTE decreased hepatic glucose production by downregulating phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) and increasing protein kinase B and AMP-activated protein kinase phosphorylation in HepG2 cells. These results correlated with the antihyperglycemic, antitriglyceridemic, anti-insulin resistance, and antioxidant activities of TTE in T2DM rats, similar to the metformin and combination treatments. Consistently, impairment of hepatic gluconeogenesis in T2DM rats was restored after single and combined treatments by reducing PEPCK and G6Pase genes. Collectively, TTE could potentially be developed as a nutraceutical product to prevent glucose overproduction in patients with obesity, insulin resistance, and diabetes who are being treated with antidiabetic drugs.

Determination of whether morphometric analysis of vertebrae in the domestic cat (Felis catus) is related to sex or skull shape.

In many mammals, gender and skull shape are related to the bone morphology of the entire body; however, this has not been well established in the domestic cat (Felis catus). This study aims to find a relationship between cervical, thoracic and lumbar vertebrae morphometrics with regard to the sex and skull shape of domestic cats. A total of 79 samples obtained from 92 dried bones of domestic cats were used to determine morphometric measurements for a total of 29 parameters. Hierarchical clustering was used to cluster the vertebral bones found in three groups: C3-T1, T2-T11 and T12-L7. The skull shape identification process employed discriminative analysis and revealed the highest training data accuracy rate at up to 86.20% in T4 followed by L1 (86.04%) Axis (85.71%) and C5 (85.18%). Sex identification employed discriminative analysis and displayed the highest training data accuracy rate at up to 75.58% in L1 followed by, T7 (71.87%) and C6 (71.79%). Moreover, we found that 14% of the samples had one vertebra missing (T13 or L1). In conclusion, domestic cat vertebral morphometrics were found to be more related to skull shape than gender. In addition, bone clustering employed morphometric data and yielded a result that was similar to that of traditional cluster analysis involving body regions.

Spirogyra neglecta Aqueous Extract Attenuates LPS-Induced Renal Inflammation.

Spirogyra neglecta (SN), commonly named "Tao" in Thai, is a genus of filamentous green macroalgae. SN contains polyphenols such as isoquercetin, catechin, hydroquinone and kaempferol. These constituents exhibit beneficial effects including anti-oxidant, anti-gastric ulcer, anti-hyperglycaemia and anti-hyperlipidaemia in both in vitro and in vivo models. Whether SN extract (SNE) has an anti-inflammatory effect in vivo remains unclear. This study examined the effect of SNE on renal function and renal organic transport in lipopolysaccharide (LPS)-induced renal inflammation in rats. Rats were randomised and divided into normal saline (NS), NS supplemented with 1000 mg/kg body weight (BW) of SNE (NS + SNE), intraperitoneally injected with 12 mg/kg BW of LPS and LPS treated with 1000 mg/kg BW of SNE (LPS + SNE). Biochemical parameters in serum and urine, lipid peroxidation concentration, kidney function and renal organic anion and cation transports were determined. LPS-injected rats developed renal injury and inflammation by increasing urine microalbumin, total malondialdehyde (MDA) and inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß protein expression, respectively. In addition, uptake of renal organic anion, [3H]-oestrone sulphate (ES), was reduced in LPS-injected rats together with increased expression of organic anion transporter 3 (Oat3). However, the renal injury and inflammation, as well as impaired Oat3 function and protein expression, were restored in LPS + SNE rats. Accordingly, SNE could be developed as nutraceutical product to prevent inflammation-induced nephrotoxicity.

Differences in osteon structure histomorphometry between puppyhood and adult stages in the Golden Retriever.

Osteon structure has been widely studied in mammals, but osteon structure in dogs has received relatively little attention, especially in terms of whether aging has any effect on osteon structure. The aim of this study was to compare the osteon structure of both flat (scapula and os coxae) and long bones (humerus, radius, ulna, metacarpus, femur and tibia) of male puppy and adult Golden Retrievers. We examined five parameters: Haversian canal diameter, Haversian canal area, osteon diameter, osteon area, and number of lacunae per osteon. Our results show that the values for Haversian canal diameter were significantly higher in the os coxae and tibia, but significantly lower in the femur of adult dogs as compared to those of puppies. The Haversian canal diameter of the other bones investigated did not show any significant differences between puppies and adult dogs. The Haversian canal area was significantly greater in the os coxae, radius and femur of adult dogs than in those of puppies. The osteon diameter and area of every bone examined were significantly smaller in puppies than in adult dogs. Lastly, the number of lacunae per osteon showed the same trend as osteon diameter and area. Plexiform bone could be found in three bones in puppies, i.e. the femur, humerus and tibia. Overall, the results of this study should provide basic knowledge on the microanatomy of cortical bone in dogs and on the possible influence age.

Synergistic effect of atorvastatin and cyanidin-3-glucoside against angiotensin II-mediated vascular smooth muscle cell proliferation and migration through MAPK and PI3K/Akt pathways.

This study aimed to investigate the mechanism of cyanidin-3-glucoside (C3G) in synergy with atorvastatin, even when it is used in low concentrations. Human aortic smooth muscle cells (HASMCs) were used to verify the synergistic mechanism of atorvastatin and C3G against angiotensin II-induced proliferation and migration. BrdU incorporation assay was used to evaluate cell proliferation. Wound healing and Boyden chamber assays were used to investigate cell migration. The cell cycle was examined using flow cytometry. The results revealed that atorvastatin and C3G exhibit a synergistic effect in ameliorating HASMC proliferation and migration by enhancing cell cycle arrest. In addition, these effects also decreased mitogen-activated protein kinase (MAPK) activity by attenuating the expression of phospho-p38, phospho-extracellular signaling-regulated kinase 1/2, and phospho-c-Jun N-terminal kinase. Furthermore, the combination of atorvastatin and C3G modulated the PI3K/Akt pathway and upregulated p21Cip1, which was associated with decreases in cyclin D1 and phospho-retinoblastoma expressions. The synergistic effect of atorvastatin and C3G induced anti-proliferation and anti-migration through MAPK and PI3K/Akt pathways mediated by AT1R. These results suggest that the synergistic effect of atorvastatin and C3G may be an alternative therapy for atherosclerosis patients.

Differences in compact bone tissue microscopic structure between adult humans (Homo sapiens) and Assam macaques (Macaca assamensis).

This study investigated the osteon structure of adult humans and Assam macaques, which served as a nonhuman primate model, to find an adequate key for species identification. Samples of compact bone from humans (n=5) and Assam macaques (n=5) - including humerus (n=20), radius (n=20), ulna (n=20), femur (n=20), tibia (n=20) and fibula (n=20) - were processed using conventional histological techniques. 100 secondary osteons from each sample were evaluated under light microscopy. Parameter measurements included: diameter, perimeter and area of Haversian canal and osteon; distance between centers of Haversian canals; and ratio between diameter of Haversian canal and osteon. Four parameters, including diameters and areas of Haversian canal and osteon, demonstrated significantly higher (P<0.05) values in humans than in Assam macaques. Therefore, compact bone microstructure could thus be used as a potential tool to differentiate human and nonhuman primates.

Saposin-like protein 2 has an immunodiagnostic potential for detecting Fasciolosis gigantica.

Saposin-like protein 2 (SAP-2) plays an important role in the digestive process of Fasciola gigantica (Fg). It is one of the major proteins synthesized by the caecal epithelial cells and released into fluke's excretion-secretion. Therefore, FgSAP-2 is a plausible target for detecting fasciolosis. A polyclonal antibody (PoAb) against recombinant FgSAP-2 was produced by immunizing rabbits with the recombinant protein (rFgSAP-2), and used in sandwich ELISA assay to detect the circulating FgSAP-2 in sera of mice experimentally infected with F. gigantica metacercariae. The assay could detect rFgSAP-2 and the native FgSAP-2 in the excretory-secretory (ES) and whole body (WB) fractions of adult F. gigantica at the concentrations as low as 38 pg/ml, 24 ng/ml, and 102 ng/ml, respectively. As well, the sera from mice experimentally infected with F. gigantica were tested positive by this sandwich ELISA, which exhibited sensitivity, specificity, false positive rate, false negative rate and accuracy at 99.99, 98.67, 1.33, 0.01 and 99.32%, respectively. Therefore, this assay could be used for diagnosis of fasciolosis by F. gigantica.

Vaccine potential of recombinant cathepsin B against Fasciola gigantica.

In Fasciola gigantica, cathepsin Bs, especially cathepsin B2 and B3 are expressed in early juvenile stages, and are proposed to mediate the invasion of host tissues. Thus they are thought to be the target vaccine candidates that can block the invasion and migration of the juvenile parasite. To evaluate their vaccine potential, the recombinant cathepsin B2 (rFgCatB2) and cathepsin B3 (rFgCatB3) were expressed in yeast, Pichia pastoris, and used to immunize mice in combination with Freund's adjuvant to evaluate the protection against the infection by F. gigantica metacercariae, and the induction of immune responses. Mice immunized with both recombinant proteins exhibited high percent of parasite reduction at 60% for rFgCatB2 and 66% for rFgCatB3. Immunization by both antigens induced continuously increasing levels of IgG1 and IgG2a with a higher level of IgG1 isotype, indicating the mixed Th1/Th2 responses with Th2 predominating. When examined individually, the higher levels of IgG1 and IgG2a were correlated with the lower numbers of worm recoveries. Thus, both cathepsin B2 and cathepsin B3 are plausible vaccine candidates whose potential should be further tested in large economic animals.

Characterization and expression of cathepsin B2 in Fasciola gigantica.

Fasciola gigantica cathepsin B belongs to a family of cysteine proteases which is involved in invasion of host tissues. In this study, the recombinant cathepsin B2 (rFgCatB2), synthesized in Pichia pastoris, showed enzymatic activity on a fluorometric substrate Z-Phe-Arg-AMC and gelatin. Furthermore, this recombinant enzyme could degrade IgG and type I collagen. Mouse antiserum against rFgCatB2 reacted with the native FgCatB2 in whole body (WB) extracts of metacercariae (MET), newly excysted juveniles (NEJ) and 2week-old juveniles, but not in 3, 4 week-old juveniles and adult flukes. Immunolocalization showed the presence of cathepsin B2 only in the caecal epithelium of MET, NEJ and 2 week-old juveniles. Co-localization of FgCatB2 and a prominent antigen of NEJ, FgCatB3, revealed that these proteins were expressed at the same regions in the caecal epithelium. Anti-rFgCatB2 showed no cross reaction with the other parasites' antigens by Western blotting. These findings suggest that CatB2 is expressed only in early stages of the parasite and may be involved in digestion of host connective tissues and evasion of the host immune system during their penetration and migration. Thus, CatB2 could be considered as an immunodiagnostic and vaccine candidate for fasciolosis.