Developmental adcyap1b loss leads to hemorrhage, disrupted hemostasis, and a blood coagulation cascade in zebrafish.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide is a neuropeptide with diverse roles in biological processes. Its involvement in the blood coagulation cascade is unclear. OBJECTIVES: This study unraveled adcyap1b's role in blood coagulation using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 in zebrafish. Effects were validated via adcyap1b knockdown. Gene expression changes in adcyap1b mutants were explored, linking them to clotting disorders. An analysis of proca gene splicing illuminated its role in adcyap1b-related anticoagulation deficiencies. METHODS: Zebrafish were genetically modified using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to induce adcyap1b knockout. Morpholino-mediated gene knockdown was employed for validation. Expression levels of coagulation factors, anticoagulant proteins, and fibrinolytic system genes were assessed in adcyap1b mutant zebrafish. Alternative splicing of proca gene was analyzed. RESULTS: Adcyap1b mutant zebrafish exhibited severe hemorrhage, clotting disorders, and disrupted blood coagulation. Morpholino-mediated knockdown replicated observed phenotypes. Downregulation in transcripts related to coagulation factors V and IX, anticoagulation protein C, and plasminogen was observed. Abnormal alternative splicing of the proca gene was identified, providing a mechanistic explanation for anticoagulation system deficiencies. CONCLUSION: Adcyap1b plays a crucial role in maintaining zebrafish blood coagulation and hemostasis. Its influence extends to the regulation of procoagulant and anticoagulant pathways, with abnormal alternative splicing contributing to observed deficiencies. These findings unveil a novel aspect of adcyap1b function, offering potential insights into similar processes in mammalian systems.

Benefits of Huang Lian mediated by gut microbiota on HFD/STZ-induced type 2 diabetes mellitus in mice.

Background: Huang Lian (HL), one of the traditional Chinese medicines (TCMs) that contains multiple active components including berberine (BBR), has been used to treat symptoms associated with diabetes for thousands of years. Compared to the monomer of BBR, HL exerts a better glucose-lowering activity and plays different roles in regulating gut microbiota. However, it remains unclear what role the gut microbiota plays in the anti-diabetic activity of HL. Methods: In this study, a type 2 diabetes mellitus (T2DM) mouse model was induced with a six-week high-fat diet (HFD) and a one-time injection of streptozotocin (STZ, 75 mg/kg). One group of these mice was administrated HL (50 mg/kg) through oral gavage two weeks after HFD feeding commenced and continued for four weeks; the other mice were given distilled water as disease control. Comprehensive analyses of physiological indices related to glycolipid metabolism, gut microbiota, untargeted metabolome, and hepatic genes expression, function prediction by PICRUSt2 were performed to identify potential mechanism. Results: We found that HL, in addition to decreasing body fat accumulation, effectively improved insulin resistance by stimulating the hepatic insulin-mediated signaling pathway. In comparison with the control group, HL treatment constructed a distinct gut microbiota and bile acid (BA) profile. The HL-treated microbiota was dominated by bacteria belonging to Bacteroides and the Clostridium innocuum group, which were associated with BA metabolism. Based on the correlation analysis, the altered BAs were closely correlated with the improvement of T2DM-related markers. Conclusion: These results indicated that the anti-diabetic activity of HL was achieved, at least partly, by regulating the structure of the gut microbiota and the composition of BAs.

Calenduloside E protects against myocardial ischemia-reperfusion injury induced calcium overload by enhancing autophagy and inhibiting L-type Ca2+ channels through BAG3.

Calenduloside E (CE) is a saponin isolated from Aralia elata (Miq) Seem, which has anti-cardiovascular disease effects. This study aims to evaluate the anti-myocardial ischemia-reperfusion injury (MIRI) mechanisms of CE and regulation of BAG3 on calcium overload. We adopted siRNA to interfere with BAG3 expression in H9c2 cardiomyocytes and used adenovirus to interfere with BAG3 expression (Ad-BAG3) in primary neonatal rat cardiomyocytes (PNRCMs) to clarify the role of BAG3 in mitigating MIRI by CE. The results showed that CE reduced calcium overload, and Ad-BAG3 had a significant regulatory effect on L-type Ca2+ channels (LTCC) but no effects on other calcium-related proteins. And BAG3 and LTCC were colocalized in myocardial tissue and BAG3 inhibited LTCC expression. Surprisingly, CE had no regulatory effect on LTCC mRNA, but CE promoted LTCC degradation through the autophagy-lysosomal pathway rather than the ubiquitination-protease pathway. Autophagy inhibitor played a negative regulation of cardiomyocyte contraction rhythm and field potential signals. Ad-BAG3 inhibited autophagy by regulating the expression of autophagy-related proteins and autophagy agonist treatment suppressed calcium overload. Therefore, CE promoted autophagy through BAG3, thereby regulating LTCC expression, inhibiting calcium overload, and ultimately reducing MIRI.

Effects of notoginseng leaf triterpenes on small molecule metabolism after cerebral ischemia/reperfusion injury assessed using MALDI-MS imaging.

BACKGROUND: Notoginseng leaf triterpenes (PNGL) is believed to have neuroprotective effects via the inhibition of inflammatory response and neuronal apoptosis. However, its mechanisms underlying the anti-ischemia/reperfusion (I/R) injury effects on the regulation of small molecule metabolism in rat brain remains unclear. The purpose of this study was thus to explore the mechanisms of PNGL on the regulation of small molecule metabolism in rat brain after I/R injury using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI). METHODS: As a model of in vivo cerebral I/R injury, male Sprague-Dawley (SD) rats were established with a middle cerebral artery occlusion/reperfusion (MCAO/R) model after PNGL administration with 40 mg·kg-1 through intraperitoneal injection (i.p.) for 7 days. We assessed the neurological behavior, regional cerebral blood flow (r CBF), neuron injury, and spatial distribution of metabolic small molecules. RESULTS: Our in vivo results suggested that PNGL increased cerebral blood flow and relieved neurological dysfunction. Furthermore, using MALDI-MSI, we demonstrated that PNGL regulated 16 endogenous small molecules implicated in metabolic networks including tricarboxylic acid (TCA) cycle, adenosine triphosphate (ATP) metabolism, malate-aspartate shuttle, metal ions, and antioxidants underwent noticeable changes after reperfusion for 24 h. CONCLUSIONS: PNGL is a novel cerebrovascular agent that can improve cerebral blood flow and attenuate adverse neurological disorders. The mechanisms are closely correlated with relative metabolic pathways, which offers insight into exploring new mechanisms in PNGL for the treatment of cerebral I/R injury.

Notoginseng Leaf Triterpenes Ameliorates OGD/R-Induced Neuronal Injury via SIRT1/2/3-Foxo3a-MnSOD/PGC-1α Signaling Pathways Mediated by the NAMPT-NAD Pathway.

BACKGROUND: Cerebral ischemic stroke (CIS) is a common cerebrovascular disease whose main risks include necrosis, apoptosis, and cerebral infarction. But few therapeutic advances and prominent drugs seem to be of value for ischemic stroke in the clinic yet. In the previous study, notoginseng leaf triterpenes (PNGL) from Panax notoginseng stem and leaf have been confirmed to have neuroprotective effects against mitochondrial damages caused by cerebral ischemia in vivo. However, the potential mechanisms of mitochondrial protection have not been fully elaborated yet. METHODS: The oxygen and glucose deprivation and reperfusion (OGD/R)-induced SH-SY5Y cells were adopted to explore the neuroprotective effects and the potential mechanisms of PNGL in vitro. Cellular cytotoxicity was measured by MTT, viable mitochondrial staining, and antioxidant marker detection in vitro.Mitochondrial functions were analyzed by ATP content measurement, MMP determination, ROS, NAD, and NADH kit in vitro. And the inhibitor FK866 was adopted to verify the regulation of PNGL on the target NAMPT and its key downstream. RESULTS: In OGD/R models, treatment with PNGL strikingly alleviated ischemia injury, obviously preserved redox balance and excessive oxidative stress, inhibited mitochondrial damage, markedly alleviated energy metabolism dysfunction, improved neuronal mitochondrial functions, obviously reduced neuronal loss and apoptosis in vitro, and thus notedly raised neuronal survival under ischemia and hypoxia. Meanwhile, PNGL markedly increased the expression of nicotinamide phosphoribosyltransferase (NAMPT) in the ischemic regions and OGD/R-induced SH-SY5Y cells and regulated the downstream SIRT1/2-Foxo3a and SIRT1/3-MnSOD/PGC-1α pathways. And FK866 further verified that the protective effects of PNGL might be mediated by the NAMPT in vitro. CONCLUSIONS: The mitochondrial protective effects of PNGL are, at least partly, mediated via the NAMPT-NAD+ and its downstream SIRT1/2/3-Foxo3a-MnSOD/PGC-1α signaling pathways. PNGL, as a new drug candidate, has a pivotal role in mitochondrial homeostasis and energy metabolism therapy via NAMPT against OGD-induced SH-SY5Y cell injury.

Total Saponins of Aralia Elata (Miq) Seem Alleviate Calcium Homeostasis Imbalance and Endoplasmic Reticulum Stress-Related Apoptosis Induced by Myocardial Ischemia/Reperfusion Injury.

BACKGROUND/AIMS: Total saponins of Aralia elata (Miq) Seem (AS) from the Chinese traditional herb Long ya Aralia chinensis L. reportedly provide cardioprotective effects, but the exact mechanisms require further study. Previous studies have showed that myocardial ischemia/ reperfusion injury (MIRI) was related to calcium homeostasis imbalance and endoplasmic reticulum stress (ERS). Thus, this study aimed to demonstrate protective effects of AS on MIRI. METHODS: After administrating AS for 5 days, the left anterior descending artery coronary artery of Sprague-Dawley (SD) rats was ligated for 30 min. After 48 h of reperfusion, haemodynamics, Evans blue/ 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (HE) staining, masson staining and the levels of lactate dehydrogenase (LDH) and creatine kinase (CK), superoxide dismutase (SOD), malondialdehyde (MDA) were detected to assess MIRI. ATPase activity and Western Blot were used to study the mechanisms. RESULTS: Compared with IR group, AS treatment groups could significantly reduce myocardial infarct size; improve myocardial pathologic progress; decrease content of LDH, CK, and MDA; increase content of SOD; and restore the activities of Ca2+-Mg2+-ATPase, Na+-K+-ATPase, sarcoplasmic reticulum Ca2+-ATPases (SERCA), and calcineurin (CaN). AS treatment groups also significantly up-regulated the expression of GRP78, C/EBP homologous protein (CHOP), and Bax, and down-regulated the expression of Bcl-2, all similar to the effects of ERS. CONCLUSION: These findings illustrated that AS could prevent myocardial ischemia/reperfusion injury and reduce calcium homeostasis imbalance and ERS-related apoptosis.

D-dencichine Regulates Thrombopoiesis by Promoting Megakaryocyte Adhesion, Migration and Proplatelet Formation.

Life-threatening chemotherapy-induced thrombocytopenia can increase the risk of bleeding due to a dramatic low platelet count, which may limit or delay treatment schedules in cancer patients. The pressing need for the rapid alleviation of the symptoms of thrombocytopenia has prompted us to search for novel highly effective and safe thrombopoietic agents. Pharmacological investigations have indicated that dencichine can prevent and treat blood loss and increase the number of platelets. On the basis of the neurotoxicity of dencichine, D-dencichine is artificially synthesized in the laboratory. Our initial results showed that D-dencichine had potential to elevate peripheral platelet levels in mice with carboplatin-induced thrombocytopenia. However, the mechanisms of D-dencichine on thrombopoiesis have been poorly understood. In this study, we found that sequential administration of D-dencichine had a distinct ability to elevate numbers of reticulated platelets, and did not alter their clearance. Moreover, we demonstrated that D-dencichine was able to modulate the return of hematopoietic factors to normal levels, including thrombopoietin and IL-6. However, subsequent analysis revealed that D-dencichine treatment had no direct effects on megakaryocytes proliferation, differentiation, and polyploidization. Further in vitro studies, we demonstrated for the first time that D-dencichine significantly stimulated megakaryocyte adhesion, migration, and proplatelet formation in a dose-dependent manner through extracellular regulated protein kinases1/2 (ERK1/2) and v-akt murine thymoma viral oncogene homolog (AKT) signaling pathways. This study sufficiently characterized the role of the effects of D-dencichine treatment on the regulation of thrombopoiesis and provided a promising avenue for CIT treating.

Oxidative stress suppression by luteolin-induced heme oxygenase-1 expression.

Luteolin, a flavonoid that exhibits antioxidative properties, exerts myocardial protection effects. However, the underlying molecular mechanisms are not yet fully understood. To investigate the effects of luteolin on myocardial injury protection and its possible mechanisms, a myocardial injury model was established with intragastric administration of 4 mg/kg isoproterenol (ISO) to male Sprague-Dawley rats (200-220 g) daily for 2 days. We found that pretreatment of luteolin (160, 80 and 40 mg/kg, i.g., respectively) daily for 15 days can prevent ISO-induced myocardial damage, including decrease of serum cardiac enzymes, improvement electrocardiography and heart vacuolation. Luteolin also improved the free radical scavenging and antioxidant potential, suggesting one possible mechanism of luteolin-induced cardio-protection is mediated by blocking the oxidative stress. To clarify the mechanisms, we performed the in vitro study by hydrogen peroxide (H(2)O(2))-induced cytotoxicty model in H9c2 cells. We found that luteolin pretreatment prevented apoptosis, increased the expression of heme oxygenase-1 (HO-1), and enhanced the binding of Nrf2 to the antioxidant response element, providing an adaptive survival response against H(2)O(2)-derived oxidative cytotoxicity. The addition of Znpp, a selective HO-1 competitive inhibitor, reduced the cytoprotective ability of luteolin, indicating the vital role of HO-1 on these effects. Luteolin also activated Akt and ERK, whereas the addition of LY294002 and U0126, the pharmacologic inhibitors of PI3K and ERK, attenuated luteolin-induced HO-1 expression and cytoprotective effect. Taken together, the above findings suggest that luteolin protects against myocardial injury and enhances cellular antioxidant defense capacity through the activation of Akt and ERK signal pathways that leads to Nrf2 activation, and subsequently HO-1 induction.