Dihydroartemisinin enhances the inhibitory effect of sorafenib on HepG2 cells by inducing ferroptosis and inhibiting energy metabolism.

Although sorafenib (Sora) shows improved efficacy in clinical liver cancer therapy, its therapeutic efficacy is still greatly limited due to side effects as well as drug resistance. Thus new drug intervention strategies are imperative. Our research showed the combined application of Dihydroartemisinin (DHA) and Sora had a synergistic inhibitory effect on HepG2 and SW480 cells, and DHA enhanced Sora efficacy on xenograft tumor in nude mice. DHA and Sora significantly inhibited the cell energy metabolism by decreasing the ATP synthesis rate of oxidative phosphorylation and glycolysis rate, and induced ferroptosis by increasing the level of lipid reactive oxygen species (L-ROS), labile iron pool (LIP) as well as malondialdehyde (MDA) and decreasing the level of glutathione (GSH) in HepG2 cells. In addition, DHA and Sora significantly decreased the levels of SLC7A11 (xCT), GCLC, GPX4, and HO-1 protein in HepG2 cells. Importantly, the above-mentioned indicators changed more significantly after the combined application of DHA and Sora as compared with Sora. In conclusion, DHA and Sora had the same mechanism, and the combined application of them could have a synergistic anti-tumor effect by inducing ferroptosis and inhibiting energy metabolism in HepG2 cells.

Effects of dihydroartemisinin, a metabolite of artemisinin, on colon cancer chemoprevention and adaptive immune regulation.

BACKGROUND: Artemisinin (ART) is an anti-malaria natural compound with a moderate anticancer action. As a metabolite of ART, dihydroartemisinin (DHA) may have stronger anti-colorectal cancer (CRC) bioactivities. However, the effects of DHA and ART on CRC chemoprevention, including adaptive immune regulation, have not been systematically evaluated and compared. METHODS: Coupled with a newly-established HPLC analytical method, enteric microbiome biotransformation was conducted to identify if the DHA is a gut microbial metabolite of ART. The anti-CRC potential of these compounds was compared using two different human CRC cell lines for cell cycle arrest, apoptotic induction, and anti-inflammation activities. Naive CD4+ T cells were also obtained for testing the compounds on the differentiation of Treg, Th1 and Th17. RESULTS: Using compound extraction and analytical methods, we observed for the first time that ART completely converted into its metabolites by gut microbiome within 24 h, but no DHA was detected. Although ART did not obviously influence cancer cell growth in the concentration tested, DHA very significantly inhibited the cancer cell growth at relatively low concentrations. DHA included G2/M cell cycle arrest via upregulation of cyclin A and apoptosis. Both ART and DHA downregulated the pro-inflammatory cytokine expression. The DHA significantly promoted Treg cell proliferation, while both ART and DHA inhibited Th1 and Th17 cell differentiation. CONCLUSIONS: As a metabolite of ART, DHA possessed stronger anti-CRC activities. The DHA significantly inhibited cell growth via cell cycle arrest, apoptosis induction and anti-inflammation actions. The adaptive immune regulation is a related mechanism of actions for the observed effects.

Study towards improving artemisinin-based combination therapies.

Covering: Up to 2020 Artemisinin has made a significant contribution towards global malaria control since its initial discovery. Countless lives have been saved by this unique and miraculous molecule. In 2006, artemisinin-based combination therapies (ACTs) were recommended by the World Health Organization (WHO) as the first-line treatment for uncomplicated malaria infection and have since remained as the mainstays of the antimalarial treatment. Even so, substantial efforts to pursue better curative effects for the treatment of malaria have never ceased, particularly with regards to the circumstances surrounding the appearance of delayed clearance of malaria parasites by 3 day ACT treatments in South-East Asian countries. Strategies to further optimize artemisinin-based therapies, including synthesizing better artemisinin derivatives, developing advanced drug delivery systems, and diversifying artemisinin partner drugs, have been proposed over the past few years. Here, we provide an updated account of the continuous efforts in improving ACTs for better efficacy in curing malarial infection.

Analysis of spleen histopathology, splenocyte composition and haematological parameters in four strains of mice infected with Plasmodium berghei K173.

BACKGROUND: Malaria is a fatal disease that presents clinically as a continuum of symptoms and severity, which are determined by complex host-parasite interactions. Clearance of infection is believed to be accomplished by the spleen and mononuclear phagocytic system (MPS), independent of artemisinin treatment. The spleen filters infected red blood cells (RBCs) from circulation through immune-mediated recognition of the infected RBCs followed by phagocytosis. This study evaluated the tolerance of four different strains of mice to Plasmodium berghei strain K173 (P. berghei K173), and the differences in the role of the spleen in controlling P. berghei K173 infection. METHODS: Using different strains of mice (C57BL/6, BALB/C, ICR, and KM mice) infected with P. berghei K173, the mechanisms leading to splenomegaly, histopathology, splenocyte activation and proliferation, and their relationship to the control of parasitaemia and host mortality were examined and evaluated. RESULTS: Survival time of mice infected with P. berghei K173 varied, although the infection was uniformly lethal. Mice of the C57BL/6 strain were the most resistant, while mice of the strain ICR were the most susceptible. BALB/c and KM mice were intermediate. In the course of P. berghei K173 infection, all infected mice experienced significant splenomegaly. Parasites were observed in the red pulp at 3 days post infection (dpi) in all animals. All spleens retained late trophozoite stages as well as a fraction of earlier ring-stage parasites. The percentages of macrophages in infected C57BL/6 and KM mice were higher than uninfected mice on 8 dpi. Spleens of infected ICR and KM mice exhibited structural disorganization and remodelling. Furthermore, parasitaemia was significantly higher in KM versus C57BL/6 mice at 8 dpi. The percentages of macrophages in ICR infected mice were lower than uninfected mice, and the parasitaemia was higher than other strains. CONCLUSIONS: The results presented here demonstrate the rate of splenic mechanical filtration and that splenic macrophages are the predominant roles in controlling an individual's total parasite burden. This can influence the pathogenesis of malaria. Finally, different genetic backgrounds of mice have different splenic mechanisms for controlling malaria infection.

[Reasearch on lipid metabolism of Plasmodium and antimalarial mechanism of artemisinin].

As a unicellular organism, Plasmodium displays a panoply of lipid metabolism pathways that are seldom found together in a unicellular organism. These pathways mostly involve the Plasmodium-encoded enzymatic machinery and meet the requirements of membrane synthesis during the rapid cell growth and division throughout the life cycle. Different lipids have varied synthesis and meta-bolism pathways. For example, the major phospholipids are synthesized via CDP-diacylglycerol-dependent pathway in prokaryotes and de novo pathway in eukaryotes, and fatty acids are synthesized mainly via type Ⅱ fatty acid synthesis pathway. The available studies have demonstrated the impacts of artemisinin and its derivatives, the front-line compounds against malaria, on the lipid metabolism of Plasmodium. Therefore, this article reviewed the known lipid metabolism pathways and the effects of artemisinin and its derivatives on these pathways, aiming to deepen the understanding of lipid synthesis and metabolism in Plasmodium and provide a theoretical basis for the research on the mechanisms and drug resistance of artemisinin and other anti-malarial drugs.

Inhibitory Mechanisms of DHA/CQ on pH and Iron Homeostasis of Erythrocytic Stage Growth of Plasmodium Falciparum.

Malaria is an infectious disease caused by Plasmodium group. The mechanisms of antimalarial drugs DHA/CQ are still unclear today. The inhibitory effects (IC50) of single treatments with DHA/CQ or V-ATPase inhibitor Baf-A1 or combination treatments by DHA/CQ combined with Baf-A1 on the growth of Plasmodium falciparum strain 3D7 was investigated. Intracellular cytoplasmic pH and labile iron pool (LIP) were labeled by pH probe BCECF, AM and iron probe calcein, AM, the fluorescence of the probes was measured by FCM. The effects of low doses of DHA (0.2 nM, 0.4 nM, 0.8 nM) on gene expression of V-ATPases (vapE, vapA, vapG) located in the membrane of DV were tested by RT-qPCR. DHA combined with Baf-A1 showed a synergism effect (CI = 0.524) on the parasite growth in the concentration of IC50. Intracellular pH and irons were effected significantly by different doses of DHA/Baf-A1. Intracellular pH was decreased by CQ combined with Baf-A1 in the concentration of IC50. Intracellular LIP was increased by DHA combined with Baf-A1 in the concentration of 20 IC50. The expression of gene vapA was down-regulated by all low doses of DHA (0.2/0.4/0.8 nM) significantly (p < 0.001) and the expression of vapG/vapE were up-regulated by 0.8 nM DHA significantly (p < 0.001). Interacting with ferrous irons, affecting the DV membrane proton pumping and acidic pH or cytoplasmic irons homeostasis may be the antimalarial mechanism of DHA while CQ showed an effect on cytoplasmic pH of parasite in vitro. Lastly, this article provides us preliminary results and a new idea for antimalarial drugs combination and new potential antimalarial combination therapies.

[Research progress on heme metabolism in intraerythrocytic plasmodium].

Heme is a key metabolic factor in all life. Malaria parasite has de novo heme-biosynthetic pathway, however the growth and development of parasite depend on the hemoglobin-derived heme metabolism process during the intraerythrocytic stages, such as the ingestion and degradation of hemoglobin in the food vacuole. The hemoglobin metabolism in the food vesicles mainly includes four aspects: hemoglobin transport and intake, hemoglobin enzymolysis to produce heme, heme polymerization into malarial pigment, and heme transport via the food vacuole. The potential mechanisms of antimalarial drugs,such as chloroquine, artemisinin and atovaquone may be related to this process. The main four aspects of this metabolic process, key metabolic enzymes, effects of antimalarial drugs on the process and their potential mechanism of action would be summarized in this paper, providing ideas for rational use and mechanism exploration of similar drugs.

[Ferroptosis pathway and its intervention regulated by Chinese materia medica].

Ferroptosis is a new form of regulated cell death which is different from apoptosis, necrosis and autophagy, and results from iron-dependent lipidperoxide accumulation. Now, it is found that ferroptosis is involved in multiple physiological and pathological processes, such as cancer, arteriosclerosis, neurodegenerative diseases, diabetes, antiviral immune response, acute renal failure, hepatic and heart ischemia/reperfusion injury. On the one hand, it could be found the appropriate drugs to promote ferroptosis to clear cancer cells and virus infected cells, etc. On the other hand, we could inhibit ferroptosis to protect healthy cells. China has a wealth of traditional Chinese medicine resources. Chinese medicine contains a variety of active ingredients that regulate ferroptosis. Here, this paper reported the research of ferroptosis pathway, targets of its inducers and inhibitors that have been discovered, and the regulatory effects of the discovered Chinese herbs and its active ingredients on ferroptosis to help clinical and scientific research.

Uric acid transporters hiding in the intestine.

CONTEXT: Hyperuricaemia is known as an abnormally increased uric acid level in the blood. Although it was observed many years ago, since uric acid excretion via the intestine pathway accounted for approximately one-third of total elimination of uric acid, the molecular mechanism of 'extra-renal excretion' was poorly understood until the finding of uric acid transporters. OBJECTIVE: The objective of this study was to gather all information related to uric acid transporters in the intestine and present this information as a comprehensive and systematic review article. METHODS: A literature search was performed from various databases (e.g., Medline, Science Direct, Springer Link, etc.). The key terms included uric acid, transporter and intestine. The period for the search is from the 1950s to the present. The bibliographies of papers relating to the review subject were also searched for further relevant references. RESULTS: The uric acid transporters identified in the intestine are discussed in this review. The solute carrier (SLC) transporters include GLUT9, MCT9, NPT4, NPT homolog (NPT5) and OAT10. The ATP binding cassette (ABC) transporters include ABCG2 (BCRP), MRP2 and MRP4. Bacterial transporter YgfU is a low-affinity and high-capacity transporter for uric acid. CONCLUSION: The present review may be helpful for further our understanding of hyperuricaemia and be of value in designing future studies on novel therapeutic pathways.

[Protective effects of three phenylallyl compounds from Guizhi decoction ox-LDL-induced oxidative stress injury of human brain microvascular endothelial cells].

The main objective of this research is to observe protective effects of three phenylallyl compounds(cinnamyl alcohol,cinnamaldehyde and cinnamic acid)from Guizhi decoction against ox-LDL-induced oxidative stress injury on human brain microvascular endothelial cells(HBMEC).In this study,the toxicity and optimal protective concentration of three phenylallyl compounds from Guizhi decoction were determined by MTT assay.The HBMEC were divided into control group(DMSO),model group(ox-LDL),tert-butylhydroquinone (t-BHQ) group,cinnamyl alcohol group, cinnamaldehyde group and cinnamic acid group.The model group were treated with ox-LDL (50 mg•L⁻¹)for 24 h,other groups were separately treated with t-BHQ, cinnamyl alcohol, cinnamaldehyde and cinnamic acid of 20 µmol•L⁻¹, and exposed to ox-LDL (50 mg•L⁻¹) for 24 h at the same time.The survival rate of HBMEC was detected by MTT assay,reactive oxygen species(ROS) production of injured cells were detected using laser scanning confocal microscope (LSCM),the content of SOD, MDA, eNOS and NO in HBMEC was determined by ELISA, and the expressions of Nrf2 mRNA were detected by quantitative Real-time PCR(qRT-PCR).The results shows that oxidative stress injury of HBMEC could be induced by ox-LDL, the three phenylallyl compounds from Guizhi decoction did not affect morphology and viability of normal HBMEC.Compared with model group, the three phenylallyl compounds from Guizhi decoction could improve the above oxidative stress status and up-regulate Nrf2 mRNA expressions in injured HBMEC(P<0.05, P<0.01) .These findings suggested that the three phenylallyl compounds from Guizhi decoction have certain protective effects against ox-LDL-induced oxidative stress injury on HBMEC(cinnamaldehyde> t-BHQ> cinnamic acid>cinnamyl alcohol),the protective mechanism maybe related to regulation of antioxidant enzymes gene expression in HBMEC by Nrf2.

[TRPV1 channel-mediated thermogenesis is a common mode for the Chinese pungent-hot or pungent-warm herbs to demonstrate their natures].

To further uncover the scientific significance and molecular mechanism of the Chinese herbs with pungent hot or warm natures, endogenous and exogenous expression systems were established by isolation of dorsal root ganglion (DRG) neurons and transfection of HEK293 cells with TRPV1 channel gene separately. On this basis, the regulation action of capsaicin, one main ingredient from chili pepper, on TRPV1 channel was further explored by using confocal microscope. Besides, the three-sites one-unit technique and method were constructed based on the brown adipose tissue (BAT), anal and tail skin temperatures. Then the effect of capsaicin on mouse energy metabolism was evaluated. Both endogenous and exogenous TRPV1 channel could be activated and this action could be specifically blocked by the TRPV1 channel inhibitor capsazepine. Simultaneously, the mice's core body temperature and BAT temperature fall down and then go up, accompanied by the increase of temperature of the mice's tail skin. Promotion of the energy metabolism by activation of TRPV1 channel might be the common way for the pungent-hot (warm) herbs to demonstrate their natures.

Ruthenium-dihydroartemisinin complex: a promising new compound for colon cancer prevention via G1 cell cycle arrest, apoptotic induction, and adaptive immune regulation.

BACKGROUND: Artemisinin (ART) and its derivatives are important antimalaria agents and have received increased attention due to their broad biomedical effects, such as anticancer and anti-inflammation activities. Recently, ruthenium-derived complexes have attracted considerable attention as their anticancer potentials were observed in preclinical and clinical studies. METHODS: To explore an innovative approach in colorectal cancer (CRC) management, we synthesized ruthenium-dihydroartemisinin complex (D-Ru), a novel metal-based artemisinin derivative molecule, and investigated its anticancer, anti-inflammation, and adaptive immune regulatory properties. RESULTS: Compared with its parent compound, ART, D-Ru showed stronger antiproliferative effects on the human CRC cell lines HCT-116 and HT-29. The cancer cell inhibition of D-Ru comprised G1 cell cycle arrest via the downregulation of cyclin A and the induction of apoptosis. ART and D-Ru downregulated the expressions of pro-inflammatory cytokines IL-1ß, IL-6, and IL-8. Although ART and D-Ru did not suppress Treg cell differentiation, they significantly inhibited Th1 and Th17 cell differentiation. CONCLUSIONS: Our results demonstrated that D-Ru, a novel ruthenium complexation of ART, remarkably enhanced its parent compound's anticancer action, while the anti-inflammatory potential was not compromised. The molecular mechanisms of action of D-Ru include inhibition of cancer cell growth via cell cycle arrest, induction of apoptosis, and anti-inflammation via regulation of adaptive immunity.

[Study on scientific connotation of four herbal properties on basis of cold and hot perceptions].

The theory of herbal properties of traditional Chinese medicine (TCM) is an indispensable part of theoretical system of TCM and plays an important role in the clinical prescription and application of TCM. In this theoretical system, the theory of four herbal properties takes a core and dominant position and becomes an indispensable part of TCM natures and actions. In combination of studies and experience, this essay proposes the latest discovery in modern biology-modern scientific connotation of cold and hot herbal properties on the basis of cold and hot perceptions of organism on the basis of analysis and summary of TRP channel protein and correlation of cold and hot perceptions of organism and thermoregulation, which is an effective approach to make breakthroughs in studies on modernization drive of the theory of four herbal properties of TCM.

[Cinnamaldehyde decreases interleukin-1beta induced PGE2 production by down-regulation of mPGES-1 and COX-2 expression in mouse macrophage RAW264.7 cells].

Cinnamaldehyde was shown to have significant anti-inflammatory and anti-pyretic actions in studies from both others' and our lab. Prostaglandin E2 (PGE2) plays a key role in generation of these pathological states, while PGE, synthase-1 (mPGES-1) is one of crucial biological elements in the process of PGE2 production. And as a downstream inducible terminal prostaglandin synthase of COX-2, mPGES-1 is now regarded as a more promising novel drug target than COX-2 and is attracting more and more attention from both academia and pharmaceutical industry. The purpose of present study was to further investigate the anti-inflammatory and antipyretic molecular mechanisms of cinnamaldehyde based on the mouse macrophage cell line RAW264. 7 in vitro. The PGE2 was identified by using the method of enzyme-linked immunosorbent assay (ELISA) and the expression of COX-2 and mPGES-1 at mRNA and protein levels was detected by the Real-time PCR and Western blotting methods respectively. The experimental results suggested that cinnamaldehyde could evidently reverse the increased production of PGE2induced by IL-1beta. Moreover, the up-regulated expression levels of mPGES-1 and COX-2 were significatly decreased. Together, these results provide compelling evidence that the down-regulated actions to both the production of PGE2 as well as the expression of mPGES-I might account for, at least in part, the anti-inflammatory and anti-pyretic effects of cinnamaldehyde.

Ferroptosis plays an essential role in the antimalarial mechanism of low-dose dihydroartemisinin.

The activation of artemisinin and its derivatives (ARTs) to generate ROS and other free radicals is mainly heme- or ferrous iron-dependent. ARTs induce ferroptosis in tumor cells, although the involvement of ferroptosis in malaria remains unclear. We found that three typical inducers of ferroptosis (erastin, RSL3 and sorafenib) could effectively mimic DHA inhibition on the growth of blood-stage parasites, which exhibited synergistic or nearly additive interactions in vitro with DHA, while the combination of DHA with ferroptosis inhibitors (deferoxamine, liproxstatin-1) had an obvious antagonistic effect. DHA, similar to ferroptosis inducers, can simultaneously induce the accumulation of ferroptosis-associated cellular labile iron and lipid peroxide. However, deferoxamine and liproxstatin-1 reduced the increase in ferrous iron and lipid peroxide caused by DHA. These results suggested that ferroptosis might be an effective way to induce cell death in parasites and could be a primary mechanism by which DHA kills parasites, with almost 50% contribution at low concentrations. These results provide a new strategy for antimalarial drug screening and clinical medication guidance.

Cinnamaldehyde up-regulates the mRNA expression level of TRPV1 receptor potential ion channel protein and its function in primary rat DRG neurons in vitro.

Cinnamaldehyde (1) is a pharmacologically active ingredient isolated from cassia twig (Ramulus Cinnamomi), which is commonly used in herbal remedies to treat fever-related diseases. Both TRPV1 and TRPM8 ion channel proteins are abundantly expressed in sensory neurons, and are assumed to act as a thermosensor, with the former mediating the feeling of warmth and the latter the feeling of cold in the body. Both of them have recently been reported to be involved in thermoregulation. The purpose of this paper is to further uncover the antipyretic mechanisms of 1 by investigating its effects on the mRNA expression levels and functions of both TRPV1 and TRPM8. The results showed that 1 could up-regulate the mRNA expression levels of TRPV1 at both 37 and 39 degrees C, and its calcium-mediating function was significantly increased at 39 degrees C, all of which could not be blocked by pretreatment of the neuronal cells with ruthenium red, a general transient receptor potential (TRP) blocker, indicating that the action of 1 was achieved through a non-TRPA1 channel pathway. In conclusion, the findings in our in vitro studies might account for part of the peripheral molecular mechanisms for the antipyretic action of 1.