Sangju Cold Granule exerts anti-viral and anti-inflammatory activities against influenza A virus in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Sangju Cold Granule (SJCG) is a classical traditional Chinese medicine (TCM) prescription described in "Item Differentiation of Warm Febrile Diseases". Historically, SJCG was employed to treat respiratory illnesses. Despite its popular usage, the alleviating effect of SJCG on influenza A virus infection and its mechanisms have not been fully elucidated. AIM OF THE STUDY: Influenza is a severe respiratory disease that threatens human health. This study aims to assess the therapeutic potential of SJCG and the possible molecular mechanism underlying its activity against influenza A virus in vitro and in vivo. MATERIALS AND METHODS: Ultrahigh-performance liquid chromatography (UPLC)-Q-Exactive was used to identify the components of SJCG. The 50% cytotoxic concentration of SJCG in MDCK and A549 cells were determined using the CCK-8 assay. The activity of SJCG against influenza A virus H1N1 was evaluated in vitro using plaque reduction and progeny virus titer reduction assays. RT-qPCR was performed to obtain the expression levels of inflammatory mediators and the transcriptional regulation of RIG-I and MDA5 in H1N1-infected A549 cells. Then, the mechanism of SJCG effect on viral replication and inflammation was further explored by measuring the expressions of proteins of the RIG-I/NF-kB/IFN(I/III) signaling pathway by Western blot. The impact of SJCG was explored in vivo in an intranasally H1N1-infected BALB/c mouse pneumonia model treated with varying doses of SJCG. The protective role of SJCG in this model was evaluated by survival, body weight monitoring, lung viral titers, lung index, lung histological changes, lung inflammatory mediators, and peripheral blood leukocyte count. RESULTS: The main SJCG chemical constituents were flavonoids, carbohydrates and glycosides, amino acids, peptides, and derivatives, organic acids and derivatives, alkaloids, fatty acyls, and terpenes. The CC50 of SJCG were 24.43 mg/mL on MDCK cells and 20.54 mg/mL on A549 cells, respectively. In vitro, SJCG significantly inhibited H1N1 replication and reduced the production of TNF-α, IFN-ß, IL-6, IL-8, IL-13, IP-10, RANTES, TRAIL, and SOCS1 in infected A549 cells. Intracellularly, SJCG reduced the expression of RIG-I, MDA5, P-NF-κB P65 (P-P65), P-IκBα, P-STAT1, P-STAT2, and IRF9. In vivo, SJCG enhanced the survival rate and decreased body weight loss in H1N1-infected mice. Mice with H1N1-induced pneumonia treated with SJCG showed a lower lung viral load and lung index than untreated mice. SJCG effectively alleviated lung damage and reduced the levels of TNF-α, IFN-ß, IL-6, IP-10, RANTES, and SOCS1 in lung tissue. Moreover, SJCG significantly ameliorated H1N1-induced leukocyte changes in peripheral blood. CONCLUSIONS: SJCG significantly reduced influenza A virus and virus-mediated inflammation through inhibiting the RIG-I/NF-kB/IFN(I/III) signaling pathway. Thus, SJCG could provide an effective TCM for influenza treatment.

CcpA-Knockout Staphylococcus aureus Induces Abnormal Metabolic Phenotype via the Activation of Hepatic STAT5/PDK4 Signaling in Diabetic Mice.

Catabolite control protein A (CcpA), an important global regulatory protein, is extensively found in S. aureus. Many studies have reported that CcpA plays a pivotal role in regulating the tricarboxylic acid cycle and pathogenicity. Moreover, the CcpA-knockout Staphylococcus aureus (S. aureus) in diabetic mice, compared with the wild-type, showed a reduced colonization rate in the tissues and organs and decreased inflammatory factor expression. However, the effect of CcpA-knockout S. aureus on the host's energy metabolism in a high-glucose environment and its mechanism of action remain unclear. S. aureus, a common and major human pathogen, is increasingly found in patients with obesity and diabetes, as recent clinical data reveal. To address this issue, we generated CcpA-knockout S. aureus strains with different genetic backgrounds to conduct in-depth investigations. In vitro experiments with high-glucose-treated cells and an in vivo model study with type 1 diabetic mice were used to evaluate the unknown effect of CcpA-knockout strains on both the glucose and lipid metabolism phenotypes of the host. We found that the strains caused an abnormal metabolic phenotype in type 1 diabetic mice, particularly in reducing random and fasting blood glucose and increasing triglyceride and fatty acid contents in the serum. In a high-glucose environment, CcpA-knockout S. aureus may activate the hepatic STAT5/PDK4 pathway and affect pyruvate utilization. An abnormal metabolic phenotype was thus observed in diabetic mice. Our findings provide a better understanding of the molecular mechanism of glucose and lipid metabolism disorders in diabetic patients infected with S. aureus.

Methazolamide Attenuates the Development of Diabetic Cardiomyopathy by Promoting ß-Catenin Degradation in Type 1 Diabetic Mice.

Methazolamide (MTZ), a carbonic anhydrase inhibitor, has been shown to inhibit cardiomyocyte hypertrophy and exert a hypoglycemic effect in patients with type 2 diabetes and diabetic db/db mice. However, whether MTZ has a cardioprotective effect in the setting of diabetic cardiomyopathy is not clear. We investigated the effects of MTZ in a mouse model of streptozotocin-induced type 1 diabetes mellitus (T1DM). Diabetic mice received MTZ by intragastric gavage (10, 25, or 50 mg/kg, daily for 16 weeks). In the diabetic group, MTZ significantly reduced both random and fasting blood glucose levels and improved glucose tolerance in a dose-dependent manner. MTZ ameliorated T1DM-induced changes in cardiac morphology and dysfunction. Mechanistic analysis revealed that MTZ blunted T1DM-induced enhanced expression of ß-catenin. Similar results were observed in neonatal rat cardiomyocytes (NRCMs) and adult mouse cardiomyocytes treated with high glucose or Wnt3a (a ß-catenin activator). There was no significant change in ß-catenin mRNA levels in cardiac tissues or NRCMs. MTZ-mediated ß-catenin downregulation was recovered by MG132, a proteasome inhibitor. Immunoprecipitation and immunofluorescence analyses showed augmentation of AXIN1-ß-catenin interaction by MTZ in T1DM hearts and in NRCMs treated with Wnt3a; thus, MTZ may potentiate AXIN1-ß-catenin linkage to increase ß-catenin degradation. Overall, MTZ may alleviate cardiac hypertrophy by mediating AXIN1-ß-catenin interaction to promote degradation and inhibition of ß-catenin activity. These findings may help inform novel therapeutic strategy to prevent heart failure in patients with diabetes.

Effect of long-term treatment of Carvacrol on glucose metabolism in Streptozotocin-induced diabetic mice.

BACKGROUND: Carvacrol is a food additive with various bioactivities, including reducing the blood glucose level as well as improvement of heart function, in diabetic mice. We explored the antihyperglycemic effect of carvacrol and its effect on the key hepatic enzymes accounting for glucose metabolism. METHODS: A streptozotocin (STZ)-induced diabetes-mellitus model in mice was used. Mice were divided randomly into a control group, diabetic group, low dose carvacrol-treated diabetic group (10 mg/kg body weight [BW]), and high dose carvacrol-treated diabetic group (20 mg/kg BW). Carvacrol was injected intraperitoneally (i.p.) in each carvacrol-treated group daily for 4 weeks and 6 weeks, respectively. The level of random plasma glucose, fasting plasma glucose, and plasma insulin was determined at 4 weeks and 6 weeks after carvacrol administration. The plasma level of total cholesterol (TC), triglycerides (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and the activity of hepatic key enzymes related to glucose metabolism were determined. RESULTS: Carvacrol treatment decreased the levels of random plasma glucose and fasting plasma glucose, significantly in a dose-dependent manner. A significant improvement in glucose tolerance and a significant decrease in the plasma level of TG were observed in carvacrol-treated diabetic mice at a dose of 20 mg/kg BW compared with that in vehicle-treated diabetic mice. There was no significant difference in the plasma level of TC and insulin between vehicle-treated diabetic mice and carvacrol-treated diabetic mice. Carvacrol treatment at a dose of 20 mg/kg BW significantly reduced the plasma level of LDH but not AST, ALT, or ALP, compared with that in the vehicle-treated diabetic group. The activity of hexokinase (HK), 6-phosphofructokinase (PFK), and citrate synthetase (CS) was increased by carvacrol treatment in diabetic mice. CONCLUSIONS: Carvacrol exerted an anti-hyperglycemic effect in STZ-induced diabetic mice. This was achieved through regulating glucose metabolism by increasing the activity of the hepatic enzymes HK, PFK, and CS.

Carvacrol protects against diabetes-induced hypercontractility in the aorta through activation of the PI3K/Akt pathway.

Vascular complications induced by diabetes constitute the principal cause of morbidity and mortality in diabetic patients. It has been reported that carvacrol (CAR) possesses a wide range of biological activities. The effects of CAR on diabetes-induced vasculopathy remain unknown. In this study, diabetic mice were created by the intraperitoneal injection of streptozotocin (STZ) in male C57BL/6 J mice to investigate whether CAR provided a protective effect against diabetes-induced vasculopathy and to investigate the underlying mechanisms. We found that CAR decreased blood glucose levels in diabetic mice. Moreover, CAR ameliorated diabetes-induced aortic morphological alterations, as evidenced by an increased thickness in the intima-media width and an increased number of vascular smooth muscle cells (VSMCs) layers. Further studies revealed that CAR inhibited hypercontractility in the aortas of diabetic mice and VSMCs in response to hyperglycemia, as evidenced by the relaxation of phenylephrine(PE)-induced vasoconstriction, the decreased expression of smooth muscle (SM)-α-actin, and the increased expression of Ki67 and proliferating cell nuclear antigen (PCNA). Furthermore, the PI3K/Akt signaling pathway was inhibited in the aortas of diabetic mice and VSMCs in response to hyperglycemia, while CAR treatment activated the PI3K/Akt signaling pathway. In conclusion, our results strongly suggest that CAR plays a protective role in diabetes-induced aortic hypercontractility, possibly by activating the PI3K/Akt signaling pathway. CAR is a potential drug for the treatment of diabetic vasculopathy.