In silico prediction and a systematic toxicology-based in vivo investigation uncovering the mechanism of aquatic toxicity caused by beta-lactam antibiotics.

Recently, beta-lactam antibiotics have gained attention as significant contributors to public health and environmental issues due to their potential toxicity. Our study employed machine learning to develop a model for assessing the aquatic toxicity of beta-lactam antibiotics on zebrafish. Notably, aztreonam (AZT), a synthetic monobactam and a subclass of beta-lactam antibiotics, demonstrated developmental effects in zebrafish embryos comparable to cephalosporins, indicating a potential for toxicity. Using a systems toxicology-based approach, we identified apoptosis and metabolic disorders as the primary pathways affected by AZT and its impurity F exposure. During the administration of monobactams, we noted that ctsbb, nos2a, and dgat2, genes associated with apoptosis and the metabolic pathway, exhibited significant differential expression. Molecular docking studies were conducted to ascertain the binding affinity between monobactam compounds and their potential targets-Ctsbb, Nos2a, and Dgat2. Furthermore, our research revealed that monobactams influence pre-mRNA alternative splicing, resulting in disruptions in the expression of genes involved in hair cells, brain, spinal cord, and fin regeneration (e.g., krt4, krt5, krt17, cyt1). Notably, we observed a correlation between the levels of rpl3 and rps7 genes, both important ribosomal proteins, and the detected alternative splicing events. Overall, this study enhances our understanding of the toxicity of beta-lactam antibiotics in zebrafish by demonstrating the developmental effects of monobactams and uncovering the underlying mechanisms at the molecular level. It also identifies potential targets for further investigation into the mechanisms of toxicity and provides valuable insights for early assessment of biological toxicity associated with antibiotic pollutants.

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.

A combination of Sophora flavescens alkaloids and Panax quinquefolium saponins attenuates coxsackievirus B3­induced acute myocarditis in mice via NF­κB signaling.

Timely treatment of viral myocarditis (VMC), a form of cardiac inflammation caused by viral infections, can reduce the occurrence of dilated cardiomyopathy and sudden death. Our previous study demonstrated the anti-inflammatory and anti-fibrotic effects of KX, a combination of Sophora flavescens alkaloids and Panax quinquefolium saponins, on an autoimmune myocarditis model in vivo. The present study explored the effects of KX on coxsackievirus B3 (CVB3)-induced acute VMC in mice. Mice were randomly divided into four groups: Control, VMC, KX-high (275 mg/kg) and KX-low (138 mg/kg). Mice in the VMC, KX-high and KX-low groups received injections of CVB3 to establish the VMC model, and those in the KX-high and KX-low groups also received KX by gavage (10 ml/kg) 2 h after virus injection until euthanasia was performed on day 7 or 21. Mice in the control group received an equal KX volume of purified water. The levels of lactate dehydrogenase (LDH), creatine kinase-myocardial band (CK-MB), cardiac troponin I (cTn-I), IL-1ß, IL-6, TNF-α and high-sensitive C-reactive protein (hs-CRP) in mouse serum was measured using ELISA. Myocardial tissue structure and degree of injury were observed using hematoxylin and eosin staining. Western blotting and reverse transcription-quantitative PCR were performed to detect the expression levels of NF-κB pathway-related mRNA and protein in myocardial tissue. The results showed that the inflammation and myocardial damage levels of the mice in the VMC group were higher at 7 days than those at 21 days. At both 7 and 21 days, KX decreased the serum CK-MB, LDH, cTn-I, IL-6, TNF-α and hs-CRP levels, and inhibited NF-κB pathway-related mRNA and protein expression in the myocardium of mice. These findings indicated that KX may reduce the inflammatory response and attenuate the pathological damage in the acute and subacute phases of CVB3-induced VMC through the NF-κB pathway.

Notoginseng leaf triterpenes ameliorates mitochondrial oxidative injury via the NAMPT-SIRT1/2/3 signaling pathways in cerebral ischemic model rats.

BACKGROUND: Due to the interrupted blood supply in cerebral ischemic stroke (CIS), ischemic and hypoxia results in neuronal depolarization, insufficient NAD+, excessive levels of ROS, mitochondrial damages, and energy metabolism disorders, which triggers the ischemic cascades. Currently, improvement of mitochondrial functions and energy metabolism is as a vital therapeutic target and clinical strategy. Hence, it is greatly crucial to look for neuroprotective natural agents with mitochondria protection actions and explore the mediated targets for treating CIS. In the previous study, notoginseng leaf triterpenes (PNGL) from Panax notoginseng stems and leaves was demonstrated to have neuroprotective effects against cerebral ischemia/reperfusion injury. However, the potential mechanisms have been not completely elaborate. Methods: The model of middle cerebral artery occlusion and reperfusion (MCAO/R) was adopted to verify the neuroprotective effects and potential pharmacology mechanisms of PNGL in vivo. Antioxidant markers were evaluated by kit detection. Mitochondrial function was evaluated by ATP content measurement, ATPase, NAD and NADH kits. And the transmission electron microscopy (TEM) and pathological staining (H&E and Nissl) were used to detect cerebral morphological changes and mitochondrial structural damages. Western blotting, ELISA and immunofluorescence assay were utilized to explore the mitochondrial protection effects and its related mechanisms in vivo. Results: In vivo, treatment with PNGL markedly reduced excessive oxidative stress, inhibited mitochondrial injury, alleviated energy metabolism dysfunction, decreased neuronal loss and apoptosis, and thus notedly raised neuronal survival under ischemia and hypoxia. Meanwhile, PNGL significantly increased the expression of nicotinamide phosphoribosyltransferase (NAMPT) in the ischemic regions, and regulated its related downstream SIRT1/2/3-MnSOD/PGC-1α pathways. Conclusion: The study finds that the mitochondrial protective effects of PNGL are associated with the NAMPT-SIRT1/2/3-MnSOD/PGC-1α signal pathways. PNGL, as a novel candidate drug, has great application prospects for preventing and treating ischemic stroke.

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.

Can artificial ecological corridors be used for ecological restoration of cultivated land in Chinese Mollisols?

Artisficial ecological corridors (AECs) are internationally recognized as a standard method for restoring the regional ecological environment. However, the coupling relationship between AECs and soil quality has rarely been studied. Harbin, a typical mollisols region in the cold area of China, has severe soil problems and remediation is urgently needed, yet AEC research in this region is lacking. Based on the perspective of soil restoration, the construction factors of ecological corridors are quantitatively evaluated. It can predict the long-term impact of AECs already built along Harbin's Ashi River on soil chemical indices. This research studied the ecological restoration of secondary woodland, cultivated land within the ecological corridor, and cultivated land outside the influence range of the corridor under the influence of continuous recovery time and different locations in the corridor (distance from the Ashe River). Soil samples were taken from 5 plots, with a total of 161 samples, and 12 indices of soil ecological characteristics were monitored. The result are as follows: It is believed that the quality restoration of mollisols through ecological corridors has great application potential. Based on the low-cost natural restoration of ecological corridors, the highest values of total phosphorus (TP) and soil organic matter (SOM) in soil indices were detected in corridors (restored for more than 10 years). In addition, after ten years of recovery, pH and electrical conductivity (EC) in the ecological corridor returned to normal from high levels in cultivated land that far exceeded the reference values. The recovery process of mollisols mass begins to decrease, then increases, and finally reaches and exceeds the reference value of standard mollisols. The redundancy analysis of soil samples found the distance to be a key factor affecting soil total nitrogen (TN), SOM, and cation exchange capacity (CEC). Recovery time is a crucial factor affecting soil total organic carbon (SOC), pH and EC. According to the TN, SOM, and CEC mollisols indices, the ecological corridor's unilateral width is 125-150m. According to the SOC, pH, and EC indices of mollisols, the AECs should complete a natural recovery cycle of a minimum of 13 years. This study reveals the change mechanism of soil quality in mollisols area corridors based on recovery time and location. This research offer ideas and a scientific basis for worldwide governments in mollisols to formulate mollisols restoration policies.

Combination of Sophora flavescens alkaloids and Panax quinquefolium saponins modulates different stages of experimental autoimmune myocarditis via the NF-κB and TGF-ß1 pathways.

Chronic cardiac inflammation and fibrosis can progress into severe forms of cardiomyopathy. Sophora flavescens alkaloids (KuShen) have been previously reported to exert anti-inflammatory effects, whereas Panax quinquefolium saponins (XiYangShen) has been shown to alleviate cardiac fibrosis. Therefore, the potential effects of their combination (KX) on different stages of autoimmune myocarditis were investigated in the present study. Mice were randomly divided into the following four groups: Control; experimental autoimmune myocarditis (EAM); KX-High (275 mg/kg); and KX-Low (138 mg/kg). A 21-day and a 60-day EAM model was established through multi-site subcutaneous injections of cardiac myosin mixed with complete Freund's adjuvant on days 0, 7, 21 and 42. Mice in the High and Low KX groups were treated by gavage (10 ml/kg) daily from day 0 (1 day before treatment) until sacrifice (day 21 or 60). Mice in the control and EAM groups received an equivalent volume of distilled water. The levels of lactate dehydrogenase (LDH), creatine kinase-myocardial band (CK-MB), cardiac troponin I (cTn-I), IL-1ß, IL-6, TNF-α, TGF-ß1, collagen type I (Col Ⅰ) and collagen type III (Col Ⅲ) were measured by ELISA in the mouse myocardial tissues or serum. Myocardial tissue structure and extent of fibrosis were visualized using H&E and Masson's staining. Western blotting and immunohistochemistry were used to measure the expression levels NF-κB and TGF-ß1 pathway proteins in the myocardial tissues. The degree of inflammation in the 21-day EAM model was found to be significantly higher compared with that in the 60-day EAM model. KX significantly reduced the inflammatory response at 21 days by decreasing the expression levels of CK-MB, LDH, cTn-I, IL-1ß, IL-6, TNF-α and TGF-ß-activated kinase 1-binding protein 1/NF-κB pathway proteins. Myocardial fibrosis in the 60-day EAM model was also significantly worse compared with that in the 21-day EAM model. However, fibrosis was significantly delayed by treatment with KX. In addition, KX significantly decreased the expression levels of TGF-ß1, Smad2, Smad4, Col I and Col III. Therefore, these data suggest that KX is beneficial for treating myocarditis by targeting multiple pathways.

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.

A systems toxicology approach to explore toxicological mechanisms of fluoroquinolones-induced testis injury.

The widespread use of fluoroquinolones (FQs) causes a serious risk to the environment and human health. Here, we evaluated the potential effect to induce testis damage by gatifloxacin (GAT) intragastrically treatment in mice (25, 50, and 100 mg/kg body weight per day for 7 days). We observed testicular weight, serum testosterone, antioxidant enzyme activity, and mRNA levels and pathways. Testicular histopathology indicated that GAT administration induced a dose-dependent spermatogenesis abnormality. At 50 mg/kg, GAT altered gene expression but did not change the weight and the levels of testosterone and antioxidant enzymes. These findings indicate that mRNA levels are more sensitive than weight and testosterone for detecting GAT testicular toxicity. We also found that GAT induced testicular damage by regulating the candidate genes associated with spermatogenesis, germ cell movement, testicular fibrosis, and reproductive axis development. This study enhances our perception of the mechanism of FQs-induced testicular toxicity and environmental effects. However, the molecular mechanism needs to be further researched.

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.

Targets Fishing and Identification of Calenduloside E as Hsp90AB1: Design, Synthesis, and Evaluation of Clickable Activity-Based Probe.

Calenduloside E (CE), a natural triterpenoid compound isolated from Aralia elata, can protect against ox-LDL-induced human umbilical vein endothelial cell (HUVEC) injury in our previous reports. However, the exact targets and mechanisms of CE remain elusive. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy. Based on the previous studies of the structure-activity relationship (SAR), we introduced an alkyne moiety at the C-28 carboxylic group of CE, which kept the protective and anti-apoptosis activity. Via proteomic approach, one of the potential proteins bound to CE-P was identified as Hsp90AB1, and further verification was performed by pure recombinant Hsp90AB1 and competitive assay. These results demonstrated that CE could bind to Hsp90AB1. We also found that CE could reverse the Hsp90AB1 decrease after ox-LDL treatment. To make our results more convincing, we performed SPR analysis and the affinity kinetic assay showed that CE/CE-P could bind to Hsp90AB1 in a dose-dependent manner. Taken together, our research showed CE could probably bind to Hsp90AB1 to protect the cell injury, which might provide the basis for the further exploration of its cardiovascular protective mechanisms. For the sake of resolving this question, we designed and synthesized a clickable activity-based probe (CE-P), which could be utilized to fish the functional targets in HUVECs using a gel-based strategy.

Araloside C Prevents Hypoxia/Reoxygenation-Induced Endoplasmic Reticulum Stress via Increasing Heat Shock Protein 90 in H9c2 Cardiomyocytes.

Araloside C (AsC) is a cardioprotective triterpenoid compound that is mainly isolated from Aralia elata. This study aims to determine the effects of AsC on hypoxia-reoxygenation (H/R)-induced apoptosis in H9c2 cardiomyocytes and its underlying mechanisms. Results demonstrated that pretreatment with AsC (12.5 µM) for 12 h significantly suppressed the H/R injury in H9c2 cardiomyocytes, including improving cell viability, attenuating the LDH leakage and preventing cardiomyocyte apoptosis. AsC also inhibited H/R-induced ER stress by reducing the activation of ER stress pathways (PERK/eIF2α and ATF6), and decreasing the expression of ER stress-related apoptotic proteins (CHOP and caspase-12). Moreover, AsC greatly improved the expression level of HSP90 compared with that in the H/R group. The use of HSP90 inhibitor 17-AAG and HSP90 siRNA blocked the above suppression effect of AsC on ER stress-related apoptosis caused by H/R. Taken together, AsC could reduce H/R-induced apoptosis possibly because it attenuates ER stress-dependent apoptotic pathways by increasing HSP90 expression.

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.

Protective effects of Araloside C against myocardial ischaemia/reperfusion injury: potential involvement of heat shock protein 90.

The present study was designed to investigate whether Araloside C, one of the major triterpenoid compounds isolated from Aralia elata known to be cardioprotective, can improve heart function following ischaemia/reperfusion (I/R) injury and elucidate its underlying mechanisms. We observed that Araloside C concentration-dependently improved cardiac function and depressed oxidative stress induced by I/R. Similar protection was confirmed in isolated cardiomyocytes characterized by maintaining Ca2+ transients and cell shortening against I/R. Moreover, the potential targets of Araloside C were predicted using the DDI-CPI server and Discovery Studio software. Molecular docking analysis revealed that Araloside C could be stably docked into the ATP/ADP-binding domain of the heat shock protein 90 (Hsp90) protein via the formation of hydrogen bonds. The binding affinity of Hsp90 to Araloside C was detected using nanopore optical interferometry and yielded KD values of 29 µM. Araloside C also up-regulated the expression levels of Hsp90 and improved cell viability in hypoxia/reoxygenation-treated H9c2 cardiomyocytes, whereas the addition of 17-AAG, a pharmacologic inhibitor of Hsp90, attenuated Araloside C-induced cardioprotective effect. These findings reveal that Araloside C can efficiently attenuate myocardial I/R injury by reducing I/R-induced oxidative stress and [Ca2+ ]i overload, which was possibly related to its binding to the Hsp90 protein.

[Effects of Qizhi Jiangtang capsule on protein expressions of InsR, PI3K, GLUT2 and p-JNK in hepatic tissues of rats with type 2 diabetes].

To observe the hypoglycemic effect of Qizhi Jiangtang capsule in rats with type 2 diabetes, and investigate the preliminary mechanism of its hypoglycemic effect, type 2 diabetes rat models were established by high glucose and high fat combined with small dose of streptozotocin (STZ). After continuous administration for 6 weeks, blood glucose, and glycosylated serum protein (GSP) levels were detected in all of the animals; immunohistochemistry assay was used to detect the number of islet ß cells; Western blot assay was used to detect the protein expression levels of insulin receptor (InsR), phosphoinositide-3 kinases (PI3K), glucose transporter-2 (GLUT2) and phosphorylated Jun N-terminal kinases (p-JNK)in hepatic tissues. The results showed that Qizhi Jiangtang capsule could reduce the blood sugar and GSP levels in serum in animals with type 2 diabetes mellitus, increase the level of insulin in serum and number of islet ß cells, increase the protein expression levels of InsR, PI3K and GLUT2, and reduce the level of p-JNK protein expression. In conclusion, Qizhi Jiangtang capsule has relatively stable hypoglycemic effect, and the mechanism may be associated with increasing the number of islet ß cells and level of insulin in serum, up-regulating the protein expression levels of InsR, PI3K and GLUT2, down-regulating the level of p-JNK protein expression in hepatic tissues, and reducing the level of insulin in hepatic tissues.

[Effects of Qizhi Jiangtang capsule on dermal ulcer in type 2 diabetic rats].

The effect of Qizhi Jiangtang vapsule (QJC) on degree of dermal ulcer cicatrization in 2 type diabetic rats was studied. Except the rats for blank group, other male Wistar rats were used to establish type 2 diabetic model by feeding with high sugar and high fat diet for four weeks and intraperitonally injecting with 30 mg•kg⁻¹ streptozotocin (STZ). After that, the rats were divided into balanced groups according to blood sugar, and received corresponding drugs for treatment for 8 weeks. At the end of week 8, 2 cm diameter circular incision was done on the back of rats. After that, the rats were administered continuously for10 days. Area of ulcer surface was detected every two days. After the last administration, wound granulation tissues were cut down to conduct pathological examination and detect the expression of VEGF, PI3K, p-ERK protein in wound tissues. The results showed that compared with the model group, after application of Qizhi Jiangtang capsule (2.24 g•kg⁻¹), the wound was significantly reduced on day 6 and day 10 of wound formation; inflammation reaction on ulcer surface was significantly reduce; Qizhi Jiangtang capsule can increase VEGF expression in the wound tissues of diabetic rats, and inhibit ERK phosphorylation. It can be concluded that Qizhi Jiangtang capsule can promote skin ulcer healing for diabetes rats, and its mechanism may be related to regulating the expression of VEGA and p-ERK proteins.

[Effect of aralosides to contraction function and calcium transient of ischemia/reperfusion myocardial cells].

To discuss the protective effect of aralosides (AS) on I/R-induced rat myocardial injury. The adult rat ventricular myocyte ischemia model was established through perfusion with sodium lactate perfusate and reperfusion with Ca(2+) -containing Tyrode's solution simulation. The cell contraction and ion concentration synchronization determination system was applied to detect the effect of AS on single I/R cell contraction and Ca2+ transients. According to the findings, AS could increase resting sarcomere length, contraction amplitude, ± dL/dt(max), calcium transient amplitude and speed of post-reperfusion myocardial cells (P < 0.05, P < 0.01), and decrease in time for achieving 90.0% of maximum relaxation, time for achieving peak value, resting calcium ratio, contraction period [Ca2+] i, time for achieving 50.0% of maximum relaxation and attenuation rate of intracellular calcium transient (P < 0.05, P < 0.01). Therefore, it is suggested that AS improved the post-reperfusion cell contraction and injury of calcium homeostasis.

Elatoside C protects against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes through the reduction of endoplasmic reticulum stress partially depending on STAT3 activation.

Endoplasmic reticulum (ER) stress-induced apoptosis has been suggested to contribute to myocardial ischemia-reperfusion (I/R) injury. Elatoside C is one of the major triterpenoid compounds isolated from Aralia elata that is known to be cardioprotective. However, its effects on I/R injury to cardiac myocytes have not been clarified. This study aimed to investigate the possible protective effect of Elatoside C against hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocyte injury and its underlying mechanisms. H9c2 cardiomyocytes were subjected to H/R in the presence of Elatoside C. Our results showed that Elatoside C (25 µM) treatment provided significant protection against H/R-induced cell death, as evidenced by improved cell viability, maintained mitochondrial membrane potential, diminished mitochondrial ROS, and reduced apoptotic cardiomyocytes (P < 0.05). These changes were associated with the inhibition of ER stress-associated apoptosis markers (GRP78, CHOP, Caspase-12 and JNK), as well as the increased phosphorylation of STAT3 and an increased Bcl2/Bax ratio. Moreover, these effects of Elatoside C were prevented by the STAT3 inhibitor Stattic. Taken together, these results suggested that Elatoside C can alleviate H/R-induced cardiomyocyte apoptosis most likely by activating the STAT3 pathways and reducing ER stress-associated apoptosis.