Cardiotoxicity of (-)-borneol, (+)-borneol, and isoborneol in zebrafish embryos is associated with Na+ /K+ -ATPase and Ca2+ -ATPase inhibition.

Borneol is an example of traditional Chinese medicine widely used in Asia. There are different isomers of chiral borneol in the market, but its toxicity and effects need further study. In this study, we used zebrafish embryos to examine the effects of exposure to three isomers of borneol [(-)-borneol, (+)-borneol, and isoborneol] on heart development and the association with Na+ /K+ -ATPase from 4 h post-fertilization (4 hpf). The results showed that the three isomers of borneol increased mortality and decreased hatching rate when the zebrafish embryo developed to 72 hpf. All three isomers of borneol (0.01-1.0 mM) significantly reduced heart rate from 48 to 120 hpf and reduced the expression of genes related to Ca2+ -ATPase (cacna1ab and cacna1da) and Na+ /K+ -ATPase (atp1b2b, atp1a3b, and atp1a2). At the same time, the three isomers of borneol significantly reduced the activities of Ca2+ -ATPase and Na+ /K+ -ATPase at 0.1 to 1.0 mM. (+)-Borneol caused the most significant reduction (p < 0.05), followed by isoborneol and (-)-borneol. Na+ /K+ -ATPase was mainly expressed in otic vesicles and protonephridium. All three isomers of borneol reduced Na+ /K+ -ATPase mRNA expression, but isoborneol was the most significant (p < 0.01). Our results indicated that (+)-borneol was the least toxic of the three isomers while the isoborneol showed the most substantial toxic effect, closely related to effects on Na+ /K+ -ATPase.

Mongolian medicine in treating type 2 diabetes mellitus combined with nonalcoholic fatty liver disease via FXR/LXR-mediated P2X7R/NLRP3/NF-κB pathway activation.

Type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD) are the most problematic metabolic diseases in the world. NAFLD encompasses a spectrum of severity, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) and fibrosis, increasing the risk of cirrhosis and hepatocellular carcinoma. Importantly, NAFLD is closely linked to obesity and tightly interrelated with insulin resistance and T2DM. T2DM and NAFLD (T2DM-NAFLD) are called as the Xike Rixijing Disease and Tonglaga Indigestion Disease respectively, in Mongolian medicine. Xike Rixijing Disease maybe develop into Tonglaga Indigestion Disease. Forturnately many Mongolian medicines show efficient treatment of T2DM-NAFLD, such as Agriophyllum squarrosum, Haliyasu (dried powder of camel placenta), Digeda-4 (herbs of Lomatogonium carinthiacum, rhizomata of Coptis chinensis, ripe fruits of Gardenia jasminoides, herbs of Dianthus superbus), Guangmingyan Siwei Decoction Powder (Halite, ripe fruits of Terminalia chebula, rhizomata of Zingiber officinale, fruit clusters of Piper longum), Tonglaga-5 (ripe fruits of Punica granatum, barks of Cinnamomum cassia, ripe fruits of Amomum kravanh, fruit clusters of Piper longum, flowers of Carthamus tinctorius), Tegexidegeqi (rhizomata of Inula helenium, ripe fruits of Gardenia jasminoides, rhizomata of Platycodon grandiflorum, rhizomata of Coptis chinensis, heartwood of Caesalpinia sappan), Ligan Shiliu Bawei San (ripe fruits of Punica granatum, barks of Cinnamomum cassia, ripe fruits of Amomum kravanh, fruit clusters of Piper longum, flowers of Carthamus tinctorius, ripe fruits of Amomum tsao-ko, rhizomata of Zingiber officinale), etc. Principles of Mongolian medicine in treating diseases: by balancing "three essences or roots" and "seven elements", strengthening liver and kidney function, transporting nutrients to enhance physical strength and disease resistance, and combined with drugs for comprehensive conditioning treatment. However, their molecular mechanisms remain unclear. In this review, we prospect that Mongolian medicines might be a promising treatment for T2DM-NAFLD by activating P2X7R/NLRP3/NF-κB inflammatory pathway via lipid-sensitive nuclear receptors (i.e., FXR and LXR).

Stimulation of AMPK Prevents Diabetes-Induced Photoreceptor Cell Degeneration.

Diabetic retinopathy (DR) is a kind of severe retinal neurodegeneration. The advanced glycation end products (AGEs) affect autophagy, and mitochondrial function is involved in DR. Adenosine-activated protein kinase (AMPK) is an important metabolic sensor that can regulate energy homeostasis in cells. However, the effect of AMPK in DR is still not fully understood. In this study, we investigated the effect of AMPK on diabetes-induced photoreceptor cell degeneration. In vivo, a diabetic mouse model was established by streptozotocin (STZ) injection. Haematoxylin-eosin (HE) staining was used to observe retinal morphology and measure the thicknesses of different layers in the retina. Electroretinogram (ERG) was used to evaluate retinal function. In vitro, 661w cells were treated with AGEs with/without an AMPK agonist (metformin) or AMPK inhibitor (compound C). Flow cytometry and CCK-8 assays were used to analyse apoptosis. Mitochondrial membrane potential was analysed by JC-1. Western blotting and qRT-PCR were used to examine the expression of related proteins and genes, respectively. The wave amplitude and the thickness of the outer nuclear layer were decreased in diabetic mice. The expression of rhodopsin and opsin was also decreased in diabetic mice. In vitro, the percentage of apoptotic cells was increased, the expression of the apoptosis-related protein Bax was increased, and Bcl-2 was decreased after AGE treatment in 661w cells. The expression of the autophagy-related protein LC3 was decreased, and p62 was increased. The mitochondrial-related gene expression and membrane potential were decreased, and mitochondrial morphology was abnormal, as observed by TEM. However, AMPK stimulation ameliorated this effect. These results indicate that AMPK stimulation can delay diabetes-induced photoreceptor degeneration by regulating autophagy and mitochondrial function.

Agriophyllum oligosaccharides ameliorate hepatic injury in type 2 diabetic db/db mice targeting INS-R/IRS-2/PI3K/AKT/PPAR-γ/Glut4 signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Agriophyllum squarrosum (L.) Moq. is a traditional Mongol medicine generally used to treat diabetes. OBJECTIVE: To investigate the protective effects and potential mechanisms of Agriophyllum oligosaccharides (AOS) on liver injury in type 2 diabetic db/db mice. MATERIALS AND METHODS: The db/db mice were divided into the model group (Model), metformin group (MET), high-dose AOS group (HAOS), and low-dose AOS group (LAOS). Nondiabetic littermate control db/m mice were used as the normal control group (Control). Mice in AOS groups were treated with AOS (380 or 750 mg/kg) daily, for 8 weeks. At 8 weeks, blood samples were collected to detect lipid and enzyme parameters concerning hepatic function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLB), triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C). Random blood glucose (RBG) test, oral glucose tolerance test (OGTT), and oral maltose tolerance test (OMTT) were also conducted. Microscopy was used to observe morphological changes in the liver of AOS-treated groups. Real-time PCR was used to detect the mRNA expression, including insulin receptor substrate 2 (IRS-2), phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), peroxisome proliferator-activated receptor (PPAR)-γ, insulin receptor (INS-R), and Glut4. Furthermore, western blotting was performed to identify proteins, including phosphorylation of IRS-2 (p-IRS-2), PI3K, p-AKT, PPAR-γ, INS-R, and Glut4. Hepatic protein expression of p-IRS-2, PI3K, p-AKT, PPAR-γ, INS-R, and Glut4 was observed using immunohistochemical staining. RESULTS: AOS treatment significantly decreased RBG, OGTT, and OMTT in mice, as well as serum ALT and AST activities. AOS groups demonstrated significantly higher expressions of p-IRS-2, PI3K, PPAR-γ, p-AKT, INS-R, and Glut4 protein and IRS-2, PI3K, AKT, PPAR-γ, INS-R, and Glut4 mRNA in the liver tissue of db/db mice; the degeneration and necrosis of hepatocytes and formation of collagen fibres markedly reduced, improving the structural disorder in the liver. CONCLUSION: The results suggest that AOS could protect the liver in type 2 diabetes, in part by activating insulin in the INS-R/IRS2/PI3K/AKT/Glut4/PPAR-γ signal pathway, facilitating hepatocyte proliferation, and further reducing the blood glucose levels.

Effects of Agriophyllum squarrosum extracts on glucose metabolism in KKAy mice and the associated underlying mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Agriophyllum squarrosum (L.) Moq. is a traditional Mongol medicine commonly used in the treatment of diabetes. AIM OF THE STUDY: To examine the effects of Agriophyllum squarrosum extract (ASE) on glucose metabolism in type 2 diabetic KKAy mice, and to investigate the mechanisms underlying these effects. MATERIAL AND METHODS: KKAy mice were divided into a model control group (MCG), a low-dose Agriophyllum squarrosum extract group (LASEG), a medium-dose Agriophyllum squarrosum extract group (MASEG), a high-dose Agriophyllum squarrosum extract group (HASEG), and a metformin group (MEG). Syngeneic C57BL/6 mice were used as a normal control group (NCG). Drugs were administered to all mice by gavage for 8 weeks. Random blood glucose levels were measured in the mice at baseline and after 2, 4, and 8 weeks of treatment. Glucose tolerance was measured after 6 weeks of drug administration. After 8 weeks, glycated serum proteins (GSP) and advanced glycation end-products (AGEs) in the serum of all mice were measured. Sections of mouse liver tissues were used for periodic acid-Schiff staining (PAS) and the content of hepatic glycogen was determined. Immunohistochemistry was used to determine the effects of ASE on liver phospho-insulin receptor substrate 2 (P-IRS2) protein expression. Western blotting was used to quantify the protein expression levels of phosphatidylinositol 3-kinase (PI3K), AKT, phospho-AKT (S473) (P-AKT), glycogen synthase kinase 3ß (GSK3ß), and glucose transporters 4 (GLUT4), while PCR was used to quantify the mRNA expression levels of insulin receptor substrate 2 (IRS2), PI3K, AKT, GSK3ß, and GLUT4. RESULTS: ASE treatment decreased random blood glucose levels in type 2 diabetic KKAy mice; increased glucose tolerance; decreased serum GSP and AGEs content; increased glycogen synthesis in liver tissues; upregulated the protein expression levels of PI3K, AKT, GLUT4, and P-IRS2; downregulated the protein expression level of GSK3ß in liver tissues; upregulated the mRNA expression levels of IRS2, PI3K, AKT, and GLUT4; and downregulated the mRNA expression level of GSK3ß in liver tissues. CONCLUSION: ASE treatment may increase glucose metabolism in KKAy mice and improve glucose tolerance. The underlying mechanisms of the beneficial effects of ASE may be associated with the increase of glycogen synthesis, the inhibition of AGEs production, the upregulation of IRS2, PI3K, AKT, and GLUT4 protein and mRNA expression, and the downregulation of GSK3ß protein and mRNA expression.

Analysis on the interaction domain of VirG and apyrase by pull-down assay.

VirG is outer membrane protein of Shigella and affects the spread of Shigella. Recently it has been reported that apyrase influences the location of VirG, although the underlying mechanism remains poorly understood. The site of interaction between apyrase and VirG is the focus of our research. First we constructed recombinant plasmid pHIS-phoN2 and pS-(v1-1102, v53-758, v759-1102, v53-319, v320-507, v507-758) by denaturation-renaturation, the phoN2:kan mutant of Shigella flexneri 5a M90T by a modified version of the lambda red recombination protocol originally described by Datsenko and Wanner and the complemented strain M90TΔphoN2/pET24a(PhisphoN2). Second, the recombinant plasmid pHIS-phoN2 and the pS-(v1-1102, v53-758, v759-1102, v53-319, v320-507, v507-758) were transformed into E. coli BL21 (DE3) and induced to express the fusion proteins. Third, the fusion proteins were purified and the interaction of VirG and apyrase was identified by pull-down. Fourth, VirG was divided and the interaction site of apyrase and VirG was determined. Finally, how apyrase affects the function of VirG was analyzed by immunofluorescence. Accordingly, the results provided the data supporting the fact that apyrase combines with the α-domain of VirG to influence the function of VirG.