Allisartan isoproxil reduces mortality of stroke-prone rats and protects against cerebrovascular, cardiac, and aortic damage.

Stroke is a common cause of death and disability. Allisartan isoproxil (ALL) is a new angiotensin II receptor blocker and a new antihypertensive drug discovered and developed in China. In the present study we investigated the therapeutic effects of ALL in stroke-prone renovascular hypertensive rats (RHR-SP) and the underlying mechanisms. The model rats were generated via two-kidney two-clip (2K2C) surgery, which led to 100% of hypertension, 100% of cerebrovascular damage as well as 100% of mortality 1 year after the surgery. Administration of ALL (30 mg · kg-1 · d-1 in diet, for 55 weeks) significantly decreased stroke-related death and prolonged lifespan in RHR-SP, but the survival ALL-treated RHR-SP remained of hypertension and cardiovascular hypertrophy compared with sham-operated normal controls. In addition to cardiac, and aortic protection, ALL treatment for 10 or 12 weeks significantly reduced cerebrovascular damage incidence and scoring, along with a steady reduction of blood pressure (BP) in RHR-SP. Meanwhile, it significantly decreased serum aldosterone and malondialdehyde levels and cerebral NAD(P)H oxidase expressions in RHR-SP. We conducted 24 h continuous BP recording in conscious freely moving RHR-SP, and found that a single intragastric administration of ALL produced a long hypotensive effect lasting for at least 12 h on systolic BP. Taken together, our results in RHR-SP demonstrate that ALL can be used for stroke prevention via BP reduction and organ protection, with the molecular mechanisms related to inhibition of angiotensin-aldosterone system and oxidative stress. This study also provides a valuable scoring for evaluation of cerebrovascular damage and drug efficacy.

Cerebral organoids transplantation improves neurological motor function in rat brain injury.

BACKGROUND AND PURPOSE: Cerebral organoids (COs) have been used for studying brain development, neural disorders, and species-specific drug pharmacology and toxicology, but the potential of COs transplantation therapy for brain injury remains to be answered. METHODS: With preparation of traumatic brain injury (TBI) model of motor dysfunction, COs at 55 and 85 days (55 and 85 d-CO) were transplanted into damaged motor cortex separately to identify better transplantation donor for brain injury. Further, the feasibility, effectiveness, and underlying mechanism of COs transplantation therapy for brain injury were explored. RESULTS: 55 d-CO was demonstrated as better transplantation donor than 85 d-CO, evidenced by more neurogenesis and higher cell survival rate without aggravating apoptosis and inflammation after transplantation into damaged motor cortex. Cells from transplanted COs had the potential of multilinage differentiation to mimic in-vivo brain cortical development, support region-specific reconstruction of damaged motor cortex, form neurotransmitter-related neurons, and migrate into different brain regions along corpus callosum. Moreover, COs transplantation upregulated hippocampal neural connection proteins and neurotrophic factors. Notably, COs transplantation improved neurological motor function and reduced brain damage. CONCLUSIONS: This study revealed 55 d-CO as better transplantation donor and demonstrated the feasibility and efficacy of COs transplantation in TBI, hoping to provide first-hand preclinical evidence of COs transplantation for brain injury.

Aggravated ulcerative colitis caused by intestinal Metrnl deficiency is associated with reduced autophagy in epithelial cells.

Metrnl is a newly identified secreted protein highly expressed in the intestinal epithelium. This study aimed to explore the role and mechanism of intestinal epithelial Metrnl in ulcerative colitis. Metrnl-/- (intestinal epithelial cell-specific Metrnl knockout) mice did not display any phenotypes of colitis under basal conditions. However, under administration of 3% dextran sodium sulfate (DSS) drinking water, colitis was more severe in Metrnl-/- mice than in WT mice, as indicated by comparisons of body weight loss, the presence of occult or gross blood per rectum, stool consistency, shrinkage in the colon, intestinal damage, and serum levels of inflammatory factors. DSS-induced colitis activated autophagy in the colon. This activation was partially inhibited by intestinal epithelial Metrnl deficiency, as indicated by a decrease in Beclin-1 and LC3-II/I and an increase in p62 in DSS-treated Metrnl-/- mice compared with WT mice. These phenomena were further confirmed by observation of autophagosomes and immunofluorescence staining for LC3 in epithelial cells. The autophagy-related AMPK-mTOR-p70S6K pathway was also activated in DSS-induced colitis, and this pathway was partially blocked by intestinal epithelial Metrnl deficiency, as indicated by a decrease in AMPK phosphorylation and an increase in mTOR and p70S6K phosphorylation in DSS-treated Metrnl-/- mice compared with WT mice. Therefore, Metrnl deficiency deteriorated ulcerative colitis at least partially through inhibition of autophagy via the AMPK-mTOR-p70S6K pathway, suggesting that Metrnl is a therapeutic target for ulcerative colitis.

Cerebral Organoids Repair Ischemic Stroke Brain Injury.

Stroke is the second leading cause of death and main cause of disability worldwide, but with few effective therapies. Although stem cell-based therapy has been proposed as an exciting regenerative medicine strategy for brain injury, there are limitations. The developed cerebral organoids (COs) represent a promising transplantation source for stroke that remains to be answered. Here, we transplanted COs at 55 days and explored the feasibility in the rat middle cerebral artery occlusion (MCAO) model of stroke. COs transplantation at 6 h or even 24 h after MCAO significantly reduces brain infarct volume and improves neurological motor function. Transplanted COs show the potential of multilineage differentiation to mimic in vivo cortical development, support motor cortex region-specific reconstruction, form neurotransmitter-related neurons, and achieve synaptic connection with host brain via in situ differentiation and cell replacement in stroke. Cells from transplanted COs show extensive migration into different brain regions along corpus callosum. The mechanisms underlying COs transplantation therapy are also associated with enhanced neurogenesis, synaptic reconstruction, axonal regeneration and angiogenesis, and decreased neural apoptosis with more survival neurons after stroke. Moreover, COs transplantation promotes predominantly exogenous neurogenesis in the transplantation periphery of ipsilateral cortex and predominantly endogenous neurogenesis in the hippocampus and subventricular zone. Together, we demonstrate the efficacy and underlying mechanisms of COs transplantation in stroke. This preliminary but promising study provides first-hand preclinical evidence for COs transplantation as a potential and effective intervention for stroke treatment.

NAD replenishment with nicotinamide mononucleotide protects blood-brain barrier integrity and attenuates delayed tissue plasminogen activator-induced haemorrhagic transformation after cerebral ischaemia.

BACKGROUND AND PURPOSE: Tissue plasminogen activator (tPA) is the only approved pharmacological therapy for acute brain ischaemia; however, a major limitation of tPA is the haemorrhagic transformation that follows tPA treatment. Here, we determined whether nicotinamide mononucleotide (NMN), a key intermediate of nicotinamide adenine dinucleotide biosynthesis, affects tPA-induced haemorrhagic transformation. EXPERIMENTAL APPROACH: Middle cerebral artery occlusion (MCAO) was achieved in CD1 mice by introducing a filament to the left MCA for 5 h. When the filament was removed for reperfusion, tPA was infused via the tail vein. A single dose of NMN was injected i.p. (300 mg·kg-1 ). Mice were killed at 24 h post ischaemia, and their brains were evaluated for brain infarction, oedema, haemoglobin content, apoptosis, neuroinflammation, blood-brain barrier (BBB) permeability, the expression of tight junction proteins (TJPs) and the activity/expression of MMPs. KEY RESULTS: In the mice infused with tPA at 5 h post ischaemia, there were significant increases in mortality, brain infarction, brain oedema, brain haemoglobin level, neural apoptosis, Iba-1 staining (microglia activation) and myeloperoxidase staining (neutrophil infiltration). All these tPA-induced alterations were significantly prevented by NMN administration. Mechanistically, the delayed tPA treatment increased BBB permeability by down-regulating TJPs, including claudin-1, occludin and zonula occludens-1, and enhancing the activities and protein expression of MMP9 and MMP2. Similarly, NMN administration partly blocked these tPA-induced molecular changes. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that NMN ameliorates tPA-induced haemorrhagic transformation in brain ischaemia by maintaining the integrity of the BBB.

Autophagy Plays an Important Role in Anti-inflammatory Mechanisms Stimulated by Alpha7 Nicotinic Acetylcholine Receptor.

Alpha7 nicotinic acetylcholine receptor (α7nAChR) has been reported to alleviate neuroinflammation. Here, we aimed to determine the role of autophagy in α7nAChR-mediated inhibition of neuroinflammation and its underlying mechanism. Experimental autoimmune encephalomyelitis (EAE) mice and lipopolysaccharide-stimulated BV2 microglia were used as in vivo and in vitro models of neuroinflammation, respectively. The severity of EAE was evaluated with neurological scoring. Autophagy-related proteins (Beclin 1, LC3-II/I, p62/SQSTM1) were detected by immunoblot. Autophagosomes were observed using transmission electron microscopy and tandem fluorescent mRFP-GFP-LC3 plasmid was applied to test autophagy flux. The mRNA levels of interleukin-6 (IL-6), IL-1ß, IL-18, and tumor necrosis factor-α (TNF-α) were detected by real-time PCR. We used 3-methyladenine (3-MA) and autophagy-related gene 5 small interfering RNA (Atg5 siRNA) to block autophagy in vivo and in vitro, respectively. Activating α7nAChR with PNU282987 ameliorates EAE severity and spinal inflammatory infiltration in EAE mice. PNU282987 treatment also enhanced monocyte/microglia autophagy (Beclin 1, LC3-II/I ratio, p62/SQSTM1, colocalization of CD45- or CD68-positive cells with LC3) both in spinal cord and spleen from EAE mice. The beneficial effects of PNU282987 on EAE mice were partly abolished by 3-MA, an autophagy inhibitor. In vitro, PNU282987 treatment increased autophagy and promoted autophagy flux. Blockade of autophagy by Atg5 siRNA or bafilomycin A1 attenuated the inhibitory effect of PNU282987 on IL-6, IL-1ß, IL-18, and TNF-α mRNA. Our results demonstrate for the first time that activating α7nAChR enhances monocyte/microglia autophagy, which suppresses neuroinflammation and thus plays an alleviative role in EAE.

Diurnal Variation of the Peripheral Cholinergic Antiinflammatory Function in Mice.

AIMS: Cholinergic antiinflammatory (CAI) pathway functions importantly in inflammation via α7 nicotinic acetylcholine receptors (α7nAChR). The present work tested circadian rhythm in peripheral CAI activity and validities of CAI activity and glucocorticoids in chronotherapy for lipopolysaccharide (LPS)-induced shock. METHODS: Vesicular acetylcholine transporter (VAChT) expressed in liver and kidney was examined every 3 h in C57BL/6 mice. Proinflammatory cytokines in serum and survival time in shock were monitored after LPS injection every 3 h. Mifepristone, antagonist of glucocorticoid receptors, and methyllycaconitine (MLA), antagonist of α7nAChR, were administrated before LPS to block antiinflammatory function of endogenous glucocorticoids and acetylcholine. RESULTS: Both levels of tumor necrosis factor α, interleukin 1ß, and interleukin 6 and mortality exhibited diurnal variations with prominent peaks when LPS was given at 15:00, and the minimum mortality occurred at 00:00. Expression of VAChT increased during resting period. MLA increased serum proinflammatory cytokines slightly, but not affected survival rate. Both differences in cytokines and in survival times between LPS injection at 15:00 and 00:00 were eliminated by mifepristone, but not by MLA. CONCLUSION: Peripheral CAI pathway exerts more powerful antiinflammatory effect during resting period. Glucocorticoids appear to be efficient in chronotherapy for septic shock.

Sodium Sulfide, a Hydrogen Sulfide-Releasing Molecule, Attenuates Acute Cerebral Ischemia in Rats.

AIMS: Acute cerebral ischemia may lead to ischemic stroke, which is a major cause of death and disability worldwide. Hydrogen sulfide (H2 S) functions importantly in mammalian systems. The present work was designed to study the effect of sodium sulfide, a donor of H2 S, on acute cerebral ischemia. METHODS: Acute cerebral focal ischemia was produced by middle cerebral artery occlusion (MCAO) in Sprague-Dawley (SD) rats. Bilateral vertebral arteries and common carotid arteries were blocked to establish cerebral global ischemia in SD rats. Acute cerebral anoxia was produced by hypobaric anoxia in C57BL/6 mice and hypoxic anoxia in SD rats. Nimodipine and aspirin were set as positive control separately. RESULTS: Infarct size after MCAO was decreased by sodium sulfide. Sodium sulfide improved cerebral energy metabolism after cerebral global ischemia and prolonged survival time of animals with acute cerebral anoxia. In addition, increased cerebral blood flow and decreased cerebrovascular resistance, blood viscosity, and thrombogenesis were observed in animals treated with sodium sulfide. In cultured neurons, sodium sulfide increased cell viability and decreased cell apoptosis induced by oxygen-glucose deprivation. CONCLUSION: Sodium sulfide, a H2 S donor, presents protective effect on acute cerebral ischemia, and might be a promising therapeutic drug.

MicroRNA-124 negatively regulates LPS-induced TNF-α production in mouse macrophages by decreasing protein stability.

AIM: MicroRNAs play pivotal roles in regulation of both innate and adaptive immune responses. In the present study, we investigated the effects of microRNA-124 (miR-124) on production of the pro-inflammatory cytokine TNF-α in lipopolysaccharide (LPS)-treated mouse macrophages. METHODS: Mouse macrophage cell line RAW264.7 was stimulated with LPS (100 ng/mL). The levels of miR-124 and TNF-α mRNA were evaluated using q-PCR. ELISA and Western blotting were used to detect TNF-α protein level in cell supernatants and cells, respectively. 3'-UTR luciferase reporter assays were used to analyze the targets of miR-124. For in vivo experiments, mice were injected with LPS (30 mg/kg, ip). RESULTS: LPS stimulation significantly increased the mRNA level of miR-124 in RAW264.7 macrophages in vitro and mice in vivo. In RAW264.7 macrophages, knockdown of miR-124 with miR-124 inhibitor dose-dependently increased LPS-stimulated production of TNF-α protein and prolonged the half-life of TNF-α protein, but did not change TNF-α mRNA levels, whereas overexpression of miR-124 with miR-124 mimic produced the opposite effects. Furthermore, miR-124 was found to directly target two components of deubiquitinating enzymes: ubiquitin-specific proteases (USP) 2 and 14. Knockdown of USP2 or USP14 accelerated protein degradation of TNF-α, and abolished the effect of miR-124 on TNF-α protein stability. CONCLUSION: miR-124, targeting USP2 and USP14, negatively regulates LPS-induced TNF-α production in mouse macrophages, suggesting miR-124 as a new therapeutic target in inflammation-related diseases.

Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, anti-inflammatory and antifungal activities.

Phytochemical investigation of the 70% EtOH extract of the leaves of Alstonia scholaris afforded seven new monoterpenoid indole alkaloids: scholarisins I-VII (1-7), and three known compounds: (3R,5S,7R,15R,16R,19E)-scholarisine F (8), 3-epi-dihydro- corymine (9), and (E)-16-formyl-5α-methoxystrictamine (10). Structural elucidation of all the compounds was accomplished by spectral methods such as 1D- and 2D-NMR, IR, UV, and HRESIMS. The isolated compounds were tested in vitro for cytotoxicity against seven tumor cell lines, anti-inflammatory activities against Cox-1 and Cox-2, and antifungal potential against five species of fungi. Compounds 1, 6, and 10 exhibited significant cytotoxicities against all the tested tumor cell lines with IC50 values of less than 30 µM and selective inhibition of Cox-2 comparable with the standard drug NS-398 (>90%). Additionally, 1, 2, 3 and 8 showed antifungal activity against two fungal strains (G. pulicaris and C. nicotianae).

Sorafenib induces apoptosis of U937 cells via inhibiting WNT signal pathway / 中国实验血液学杂志

This study was aimed to investigate the effect of multikinase inhibitor sorafenib on the proliferation and apoptosis of U937 cells and its possible mechanism. U937 cells were treated with different concentrations of sorafenib for 48 hours. Cell viability was determined by Cell Counting Kit-8; cell apoptosis and cell ratio in cell cycle were detected by flow cytometry with Annexin V/PI staining and PI staining respectively; expressions of GSK-3β, β-catenin and cyclin-D1 were assayed by Western blot. The results showed that the proliferation of U937 cells was inhibited by sorafenib in a dose-dependent manner (p < 0.05). Sorafenib induced cell apoptosis and cell cycle G(1)/G(0) arrest. Compared with results of Western blot before treatment, expression of inactivated GSK-3β, β-catenin and Cyclin-D1 down-regulated in a dose-dependent manner after treatment with sorafenib, this same changes were observed after up-regulation of inactivated GSK-3β by LiCl (p < 0.05). It is concluded that sorafenib inhibits the proliferation of U937 cells and induces cell apoptosis through reducing negative regulation of WNT signal pathway on inactivated GSK-3β and down-regulating β-catenin and cyclin-D1 level, which result in U937 cell cycle G(1)/G(0) arrest.

Effect of sorafenib combined with daunorubicin on K562 cell line / 中国实验血液学杂志

The aim of this study was to investigate the effect of sorafenib combined with daunorubicin on leukemic k562 cell line. The inhibitory effect of sorafenib alone and its combination with daunorubicin on K562 cell proliferation was detected by MTT method; the synergistic effect was measured by CDI (coefficient of drug interaction); the apoptosis of K562 cells was observed by flow cytometry with Hoechst 33258 staining. The results showed that the sorafenib alone or its combination with daunorubicin could significantly inhibit K562 cell proliferation and the combination of both drugs displayed synergistic effect on K562 cells, meanwhile the apoptotic cells increased. It is concluded that the combination of sorafenib and daunorubicin has a obviously synergistic inhibitory effect on leukemic cell line K562.

Effects of Changtai granules, a traditional compound Chinese medicine, on chronic trinitrobenzene sulfonic acid-induced colitis in rats.

AIM: To study the effects of Changtai granules (CTG), a traditional compound Chinese medicine, on chronic trinitrobenzene sulfonic acid-induced colitis in rats. METHODS: Healthy adult Sprague-Dawley (SD) rats of both sexes, weighing 250-300 g, were employed in the present study. The rat colitis models were induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) enemas at a concentration of 100 mg/kg in 50% ethanol. The experimental animals were randomly divided into dexamethasone (DX) treatment, CTG treatment, and model control groups, which were intracolicly treated daily with DX (0.2 mg/kg), CTG at doses of 2.9, 5.7 and 11.4 g crude drug/kg, and the equal amount of saline respectively from 6 h following induction of the colitis in rats inflicted with TNBS to the end of study. A normal control group of rats treated without TNBS but saline enema was also included in the study. After 3 wk of treatment, the animals were assessed for colonal inflammatory and ulcerative responses with respect to mortality, frequency of diarrhea, histology and myeloperoxidase activity (MPO). RESULTS: The therapeutic effect of CTG on ulcerative colitis (UC) was better than DX. CTG effectively inhibited the activity of granulocytes, macrophages and monocytes in a dose-dependent manner. Also it reduced MPO and formation of inflammation in colonic mucosal tissue. Furthermore, administration of CTG significantly prevented body mass loss and death, and decreased frequency of diarrhea in UC rats, when compared with the model control group rats. CONCLUSION: CTG would prove to be an ideal drug for chronic UC, and is warranted to be studied further.

Inhibitors of 5-lipoxygenase inhibit expression of intercellular adhesion molecule-1 in human melanoma cells.

AIM: To study the effect of 5-lipoxygenase inhibitors on the expression of intercellular adhesion molecule-1 (ICAM-1) in melanoma cells. METHODS: ICAM-1 protein of human melanoma cell a375 was detected by enzyme-linked immunosorbent, flow cytometry and Western blot analysis. Level of ICAM-1 mRNA in a375 was evaluated by Northern blot analysis. Adhesion of a375 to endothelial cell EC304 was analyzed by isotopic tracing. RESULTS: 5-Lipoxygenase inhibitors nordihydroguaiaretic acid, AA861 and MK886, could suppress the expression of ICAM-1 protein as well as of its mRNA in a375 cells and reduce the adhesion of a375 to EC304. CONCLUSION: 5-Lipoxygenase inhibitors can inhibit the expression of ICAM-1 in human melanoma cells and may be valuable for treatment of melanoma metastasis.