ROCK1 inhibition improves wound healing in diabetes via RIPK4/AMPK pathway.

Refractory wounds are a severe complication of diabetes mellitus that often leads to amputation because of the lack of effective treatments and therapeutic targets. The pathogenesis of refractory wounds is complex, involving many types of cells. Rho-associated protein kinase-1 (ROCK1) phosphorylates a series of substrates that trigger downstream signaling pathways, affecting multiple cellular processes, including cell migration, communication, and proliferation. The present study investigated the role of ROCK1 in diabetic wound healing and molecular mechanisms. Our results showed that ROCK1 expression significantly increased in wound granulation tissues in diabetic patients, streptozotocin (STZ)-induced diabetic mice, and db/db diabetic mice. Wound healing and blood perfusion were dose-dependently improved by the ROCK1 inhibitor fasudil in diabetic mice. In endothelial cells, fasudil and ROCK1 siRNA significantly elevated the phosphorylation of adenosine monophosphate-activated protein kinase at Thr172 (pThr172-AMPKα), the activity of endothelial nitric oxide synthase (eNOS), and suppressed the levels of mitochondrial reactive oxygen species (mtROS) and nitrotyrosine formation. Experiments using integrated bioinformatics analysis and coimmunoprecipitation established that ROCK1 inhibited pThr172-AMPKα by binding to receptor-interacting serine/threonine kinase 4 (RIPK4). These results suggest that fasudil accelerated wound repair and improved angiogenesis at least partially through the ROCK1/RIPK4/AMPK pathway. Fasudil may be a potential treatment for refractory wounds in diabetic patients.

[Cardiovascular complications in malaria: a review].

Malaria, one of the major global public health events, is a leading cause of mortality and morbidity among children and adults in tropical and subtropical regions(mainly in sub-Saharan Africa), threatening human health. It is well known that malaria can cause various complications including anemia, blackwater fever, cerebral malaria, and kidney damage. Conventionally, cardiac involvement has not been listed as a common reason affecting morbidity and mortality of malaria, which may be related to ignored cases or insufficient diagnosis. However, the serious clinical consequences such as acute coronary syndrome, heart failure, and malignant arrhythmia caused by malaria have aroused great concern. At present, antimalarials are commonly used for treating malaria in clinical practice. However, inappropriate medication can increase the risk of cardiovascular diseases and cause severe consequences. This review summarized the research advances in the cardiovascular complications including acute myocardial infarction, arrhythmia, hypertension, heart failure, and myocarditis in malaria. The possible mechanisms of cardiovascular diseases caused by malaria were systematically expounded from the hypotheses of cell adhesion, inflammation and cytokines, myocardial apoptosis induced by plasmodium toxin, cardiac injury secondary to acute renal failure, and thrombosis. Furthermore, the effects of quinolines, nucleoprotein synthesis inhibitors, and artemisinin and its derivatives on cardiac structure and function were summarized. Compared with the cardiac toxicity of quinolines in antimalarial therapy, the adverse effects of artemisinin-derived drugs on heart have not been reported in clinical studies. More importantly, the artemisinin-derived drugs demonstrate favorable application prospects in the prevention and treatment of cardiovascular diseases, and are expected to play a role in the treatment of malaria patients with cardiovascular diseases. This review provides reference for the prevention and treatment of malaria-related cardiovascular complications as well as the safe application of antimalarials.

Establishment and evaluation of glucose-modified nanocomposite liposomes for the treatment of cerebral malaria.

Cerebral malaria (CM) is a life-threatening neurological complication caused by Plasmodium falciparum. About 627,000 patients died of malaria in 2020. Currently, artemisinin and its derivatives are the front-line drugs used for the treatment of cerebral malaria. However, they cannot target the brain, which decreases their effectiveness. Therefore, increasing their ability to target the brain by the nano-delivery system with brain-targeted materials is of great significance for enhancing the effects of antimalarials and reducing CM mortality. This study used glucose transporter 1 (GLUT1) on the blood-brain barrier as a target for a synthesized cholesterol-undecanoic acid-glucose conjugate. The molecular dynamics simulation found that the structural fragment of glucose in the conjugate faced the outside the phospholipid bilayers, which was conducive to the recognition of brain-targeted liposomes by GLUT1. The fluorescence intensity of the brain-targeted liposomes (na-ATS/TMP@lipoBX) in the mouse brain was significantly higher than that of the non-targeted liposomes (na-ATS/TMP@lipo) in vivo (P < 0.001) after intranasal administration. The infection and recurrence rate of the mice receiving na-ATS/TMP@lipoBX treatment were significantly decreased, which had more advantages than those of other administration groups. The analysis of pharmacokinetic data showed that na-ATS/TMP@lipoBX could enter the brain in both systemic circulation and nasal-brain pathway to treat malaria. Taken together, these results in this study provide a new approach to the treatment of cerebral malaria.

Shengmai Yin alleviated plaque vulnerability and ischemic myocardial damage in diesel exhaust particle-aggravated atherosclerosis with myocardial ischemia.

Exposure to diesel exhaust particles (DEP) increases the risk of ischemic heart disease, especially heart attacks and ischemic/thrombotic strokes. Shengmai Yin (SMY) is a traditional Chinese medicine used to treat coronary heart disease. The aim of this study was to determine the protective role of SMY and the mechanism by which SMY affects DEP-induced cardiovascular injury. This study is expected to provide the basis for the development of an adaptive signature of SMY in the prevention of atherosclerotic cardiovascular disease and premature death from global air pollution exposure. We developed animal models of myocardial ischemia and atherosclerosis (AS) in response to DEP exposure. After SMY treatment, serum lipids returned to normal. Aortic plaque area and MMP9 expression were significantly reduced and collagen fiber expression increased after SMY treatment compared to DEP exposure alone. Thus, the risk of plaque formation and vulnerability is reduced. In addition, SMY improved left ventricular structure, morphology, function, blood flow, infarct area, myocardial damage, and ROS accumulation to varying degrees in ApoE-/- mice. These results indicate that the use of SMY is effective, to varying degrees, for the treatment of dyslipidemia, atherosclerosis, myocardial ischemia, and oxidative stress in ApoE-/- mice. SMY has a potential protective effect in DEP-aggravated AS in people with myocardial ischemia.

Combined Transcriptome and Proteome Profiling for Role of pfEMP1 in Antimalarial Mechanism of Action of Dihydroartemisinin.

Malaria parasites induce morphological and biochemical changes in the membranes of parasite-infected red blood cells (iRBCs) for propagation. Artemisinin combination therapies are the first-line antiplasmodials in countries of endemicity. However, the mechanism of action of artemisinin is unclear, and drug resistance decreases long-term efficacy. To understand whether artemisinin targets or interacts with iRBC membrane proteins, this study investigated the molecular changes caused by dihydroartemisinin (DHA), an artemisinin derivative, in Plasmodium falciparum 3D7 using a combined transcriptomic and membrane proteomic profiling approach. Optical microscopy and scanning electron microscopy showed that DHA can cause morphological variation in the iRBC membrane. We identified 125 differentially expressed membrane proteins, and functional analysis indicated structural molecule activity and protein export as key biological functions of the two omics studies. DHA treatment decreased the expression of var gene variants PF3D7_0415700 and PF3D7_0900100 dose-dependently. Western blotting and immunofluorescence analysis showed that DHA treatment downregulates the var gene encoding P. falciparum erythrocyte membrane protein-1 (pfEMP1). pfEMP1 knockout significantly increased artemisinin sensitivity. Results showed that pfEMP1 might be involved in the antimalarial mechanism of action of DHA and pfEMP1 or its regulated factors may be further exploited in antiparasitic drug design. The findings are beneficial for elucidating the potential effects of DHA on iRBC membrane proteins and developing new drugs targeting iRBC membrane. IMPORTANCE Malaria parasites induce morphological and biochemical changes in the membranes of parasite-infected red blood cells (iRBCs) for propagation, with artemisinin combination therapies as the first-line treatments. To understand whether artemisinin targets or interacts with iRBC membrane proteins, this study investigated the molecular changes caused by dihydroartemisinin (DHA), an artemisinin derivative, in Plasmodium falciparum 3D7 using a combined transcriptomic and membrane proteomic profiling approach. We found that DHA can cause morphological changes of iRBC membrane. Structural molecule activity and protein export are considered to be the key biological functions based on the two omics studies. pfEMP1 might be involved in the DHA mechanism of action. pfEMP1 or its regulated factors may be further exploited in antiparasitic drug design. The findings are beneficial for elucidating the potential effects of DHA on iRBC membrane proteins and developing new antimalarial drugs targeting iRBC membrane.

Aggravation of Myocardial Ischemia upon Particulate Matter Exposure in Atherosclerosis Animal Model.

The health problems caused by air pollution (especially particulate pollution) are getting more and more attention, especially among cardiovascular disease patients, which aggravates complicated disorders and causes poor prognosis. The simple myocardial ischemia (MI) or particulate matter (PM) exposure model is unsuitable for such studies of diseases with multiple causes. Here, a method for constructing a composite model combining PM exposure, atherosclerosis, and myocardial ischemia has been described. ApoE-/- mice were fed with a high-fat diet for 16 weeks to develop atherosclerosis, tracheal instillation of PM standard suspension was performed to simulate the pulmonary exposure of PM, and the left anterior descending coronary artery was ligated one week after the last exposure. Tracheal instillation of PM can simulate acute lung exposure while significantly reducing the cost of the experiment; the classic left anterior descending artery ligation with noninvasive tracheal intubation and a new auxiliary expansion device can ensure the animal's survival rate and reduce the difficulty of the operation. This animal model can reasonably simulate the patient's pathological changes of myocardial infarction aggravated by air pollution and provide a reference for the construction of animal models related to studies involving diseases with multiple causes.

Artesunate and Tetramethylpyrazine Exert Effects on Experimental Cerebral Malaria in a Mechanism of Protein S-Nitrosylation.

Cerebral malaria (CM) is caused by Plasmodium falciparum, resulting in severe sequelae; one of its pathogenic factors is the low bioavailability of nitric oxide (NO). Our previous study suggested that the combination of artesunate (AS) and tetramethylpyrazine (TMP) exerts an adjuvant therapeutic effect on the symptoms of experimental CM (ECM) and that NO regulation plays an important role. In the present study, we further verified the effects of AS+TMP on cerebral blood flow (CBF) and detected NO-related indicators. We focused on the role of NO through S-nitrosoproteome based on previous proteomics data and explored the mechanism of AS+TMP for improving pathological ECM symptoms. We observed that AS+TMP reduces adhesion, increases CBF, and regulates NO synthase (NOS) activity, thereby regulating the level of S-nitrosothiols, such as metabolism-related or neuro-associated receptors, for improving ECM symptoms. These results demonstrated that AS+TMP could be an effective strategy in adjuvant therapy of CM.

The Protective Effects of Shengmai Formula Against Myocardial Injury Induced by Ultrafine Particulate Matter Exposure and Myocardial Ischemia are Mediated by the PI3K/AKT/p38 MAPK/Nrf2 Pathway.

Background and Purpose: Ultrafine particulate matter (UFPM) induces oxidative stress (OS) and is considered to be a risk factor of myocardial ischemia (MI). Shengmai formula (SMF) is a traditional Chinese medicine with antioxidant properties and has been used to treat cardiovascular diseases for a long time. The aim of this study was to explore the protective role of SMF and the mechanism by which it prevents myocardial injury in UFPM-exposed rats with MI. Methods: An MI rat model was established. Animals were randomly divided into five groups: sham, UFPM + MI, SMF (1.08 mg/kg⋅d) + UFPM + MI, SMF (2.16 mg/kg⋅d) + UFPM + MI, and SMF (4.32 mg/kg⋅d) + UFPM + MI. SMF or saline was administrated 7 days before UFPM instillation (100 µg/kg), followed by 24 h of ischemia. Physiological and biochemical parameters were measured, and histopathological examinations were conducted to evaluate myocardial damage. We also explored the potential mechanism of the protective role of SMF using a system pharmacology approach and an in vitro myoblast cell model with small molecule inhibitors. Results: UFPM produced myocardial injuries on myocardial infarct size; serum levels of LDH, CK-MB, and cardiac troponin; and OS responses in the rats with MI. Pretreatment with SMF significantly attenuated these damages via reversing the biomarkers. SMF also improved histopathology induced by UFPM and significantly altered the PI3K/AKT/MAPK and OS signaling pathways. The expression patterns of Cat, Gstk1, and Cyba in the UFPM model group were reversed in the SMF-treated group. In in vitro studies, SMF attenuated UFPM-induced reactive oxygen species production, mitochondrial damage, and OS responses. The PI3K/AKT/p38 MAPK/Nrf2 pathway was significantly changed in the SMF group compared with that in the UFPM group, whereas opposite results were obtained for pathway inhibition. Conclusion: These findings indicate that SMF prevents OS responses and exerts beneficial effects against myocardial injury induced by UFPM + MI in rats. Furthermore, the PI3K/AKT/p38 MAPK/Nrf2 signaling pathway might be involved in the protective effects of SMF.

Synergistic Effect of Combined Artesunate and Tetramethylpyrazine in Experimental Cerebral Malaria.

Intravenous artesunate is effective against cerebral malaria (CM), but high mortality and neurological sequelae in survivors are inevitable. We investigated the effect of combined artesunate and tetramethylpyrazine using mouse models of experimental cerebral malaria (ECM). Artesunate + tetramethylpyrazine reduced microvascular blockage and improved neurological function, including the rapid murine coma and behavior scale (RMCBS), leading to improved survival and reduced pathology in ECM. This combination downregulated the expression of adhesion molecules and sequestration of parasitized red blood cells (pRBCs), increased cerebral blood flow, nerve growth factor (b-NGF), vascular endothelial growth factor A (VEGF-A), and neurotrophin (brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3)) levels, and alleviated hippocampal neuronal damage and astrocyte activation. Down- (n = 128) and upregulated (n = 64) proteins were identified in the artesunate group, while up- (n = 217) and downregulated (n = 177) proteins were identified in the artesunate + tetramethylpyrazine group, presenting a significantly altered proteome profile. KEGG analysis showed that 166 differentially expressed proteins were enriched in the Art group and 234, in the artesunate + tetramethylpyrazine group. The neuroprotective effects of artesunate + tetramethylpyrazine were mainly related to proteins involved in axon development and transportation between blood and brain. These results suggested that artesunate + tetramethylpyrazine could be a potential adjuvant therapy against CM, but this will have to be confirmed in future studies and trials.

[Method for rapid synchronization of different growth cycles of Plasmodium falciparum in vitro and application in differential gene expression profile of 3D7 after dihydroartemisinin treatment].

Plasmodium culture in vitro is often used as an antimalarial drug evaluation model, but the lifecycle of P. falciparum culture in vitro tends to be disordered, which affects the research and evaluation of antimalarial drug mechanism in vitro. By combining magnetic bead separation method with sorbitol synchronization method, a synchronization method was constructed to quickly acquire different lifecycles of P. falciparum and obtain large amounts of parasite with a narrow synchronization window in a short period. Furthermore, the dihydroartemisinin(DHA) was used to treat the early trophozoite phase of P. falciparum 3 D7 for 4 h. Then mRNA was extracted and RNA-seq was conducted to analyze the differential expression of mRNA after drug treatment and obtain the differential gene expression profile. Differential expression of up-regulated genes and down-regulated genes was analyzed according to the screening criteria of |log_2FC|>1 and P<0.05. There, 262 genes were up-regulated and 77 genes were down-regulated. GO functional enrichment analysis of all the differentially expressed genes showed that the enrichment items mainly included cell membrane components, transporter activity, serine/threonine kinase activity, Maurer's clefts(MCs), rhoptry, antigen variation and immune evasion. The enrichment of KEGG pathway included malaria, fatty acid metabolism and peroxisome. Protein-protein interaction(PPI) analysis showed that the down-regulated genes in the modules with high degree of association included rhoptry, myosin complex, transporter and other genes related to the important life activities of malaria invasion and immune escape; the up-regulated genes were mainly related to various toxic exportins of malaria, such as PfSBP1 of MCs. qRT-PCR was used to verify the expression level of some genes, and most of the results were the same as the sequencing results. SBP1 was significantly up-regulated, while some antigenic protein expression levels were down-regulated. Above all, key molecules of DHA therapy were mainly involved in the parasites' rhoptry, transporter, antigenic variation, plasmodium exportin. These results offer us many hints to guide the further studies on mechanism of artemisinin and provide a new way for development of new antimalarial drugs.

[Protective effects of artesunate combination on experimental cerebral malaria and nerve injury].

Cerebral malaria (CM) is the leading cause of death in children under 5 years in Africa, severe neurological sequelae may occur in surviving children. Although artesunate has made breakthrough progress in the clinical treatment of CM, the clinical problems of high mortality and high morbidity have not yet been completely resolved. In this study, an experimental cerebral malaria (ECM) model was established by infecting C57BL/6 mice with Pb ANKA (Plasmodium berghei ANKA) to compare parasitemia level, survival rates, and rapid murine coma behavior scale scores, cerebral microvascular obstruction, haemozoin deposition in the liver, body temperature and weight to investigate the anti-cerebral malaria effect of the artesunate compound combination. The results showed that the artesunate compound combination could improve the survival rate of Pb ANKA-infected mice, reduce the level of parasitemia, effectively improve the symptoms of ECM neurological injury, reduce cerebrovascular obstruction and haemozoin deposition in the liver, and also significantly improve body temperature, weight and other basic indicators. The results showed that the artesunate compound combination improved the pathological changes and neurological damage caused by CM. It is expected to provide a theoretical basis for human cerebral malaria patients in clinical adjuvant therapy.

[Effect of dihydroartemisinin at low concentration on intervention of Plasmodium falciparum 3D7 strain].

Malaria is still the most severe strain of the human malaria parasites, and malaria disease is life-threatening which can result in severe anemia and cerebral malaria, especially in children in tropical Africa. Previous studies have shown that artemisinin and its derivatives could selectively kill erythrocytic stage of malaria and have a greater impact on the ring period. In recent years, there have been new findings of its mechanism continually. However, the concentration of artemisinin and its derivatives used in these studies can reach 50 to 80 times the half-inhibitory concentration in vitro. In this study, the international standard strain 3D7 of Plasmodium falciparum was used to culture in vitro. After half-inhibitory concentration of dihydroartemisinin was treated, the morphological changes of P. falciparum intraerythrocytic stage were observed, and then the 3D7 life cycle and effects of different developmental stages after dosing was explored. The 3D7 strain of P. falciparum was continuously synchronised more than 3 times. And dihydroartemisinin (DHA) at half maximal inhibitory concentration (10 nmol·L⁻¹) was administered for 6 hours after the last synchronization, and 3 life cycles were continuously observed (132 h). The results showed that compared with the parasites untreated by DHA, there was a noticeable delay in the life cycle of at least 36 h, indicating that the growth of 3D7 was significantly inhibited by DHA (P<0.001), and the rate of ring formation was significantly reduced (P<0.05). The trophozoites were abnormal in shape, such as shrink in size, and the number of merozoites in schizonts was significantly decreased (P<0.05). These results suggested that non-killing concentrations of DHA (meaning parasites can be inhibited but not killed) can significantly inhibit the growth of P. falciparum, which may not only affect the ring stage, but also have an impact on other stages of the P. falciparum.

Comparison between UV and VUV photolysis for the pre- and post-treatment of coking wastewater.

In this study, ultraviolet (UV) and vacuum ultraviolet (VUV) photolysis were investigated for the pre-treatment and post-treatment of coking wastewater. First, 6-fold diluted raw coking wastewater was irradiated by UV and VUV. It was found that 15.9%-35.4% total organic carbon (TOC) was removed after 24 hr irradiation. The irradiated effluent could be degraded by the acclimated activated sludge. Even though the VUV photolysis removed more chemical oxygen demand (COD) than UV, the UV-irradiated effluent demonstrated better biodegradability. After 4 hr UV irradiation, the biological oxygen demand BOD5/COD ratio of irradiated coking wastewater increased from 0.163 to 0.224, and its toxicity decreased to the greatest extent. Second, the biologically treated coking wastewater was irradiated by UV and VUV. Both of them were able to remove 37%-47% TOC within 8 hr irradiation. Compared to UV, VUV photolysis could significantly improve the transparency of the bio-treated effluent. VUV also reduced 7% more ammonia nitrogen (NH4+-N), 17% more nitrite nitrogen (NO2--N), and 18% more total nitrogen (TN) than UV, producing 35% less nitrite nitrogen (NO3--N) as a result. In conclusion, UV irradiation was better in improving the biodegradability of coking wastewater, while VUV was more effective at photolyzing the residual organic compounds and inorganic N-species in the bio-treated effluent.

Adsorption characteristics of ammonium exchange by zeolite and the optimal application in the tertiary treatment of coking wastewater using response surface methodology.

Natural zeolite is a favorable NH(4)(+)-ion exchanger in the tertiary wastewater treatment. In this study, a natural Chinese zeolite was anatomized using the mercury injection method, X-ray diffraction, and scanning electron micrographs. The kinetic process of ammonium adsorption onto the zeolite was best described by the pseudo second order model; the adsorption equilibrium data fitted better to the Freundlich isotherm; and the exchange between ammonium and alkali/alkaline earth cations was in the order of Na(+) > Ca(2+) > K(+) > Mg(2+). Finally, the zeolite powder was applied for the tertiary treatment of coking wastewater, which still contained high concentration of ammonium after the secondary treatment by a sequencing batch reactor. The Box-Behnken design was used to design the experimental protocol, and the response surface methodology (RSM) was used for the optimization of adsorption factors. The RSM analysis showed the optimal adsorption factors as particle size, 0.03 mm; initial dosage of zeolite powder, 50.0 g/L; and contact time, 24 h. The highest ammonium removal rate was 75.0% predicted by the RMS. Considering settleability of the zeolite powder, the particle size of 0.25 mm was recommended in practice with a little loss of the ammonium removal: 70.9% as the RMS predicted.