Effects and mechanisms of dihydroartemisinin combined with carfilzomib on the activity, proliferation, and apoptosis of multiple myeloma cells / 国际肿瘤学杂志

Objective:To study the effects and potential mechanisms of the combination of dihydroartemisinin and carfilzomib on the activity, proliferation, and apoptosis of multiple myeloma ARD cell lines.Methods:In vitro cultivation of multiple myeloma ARD cells involved treating the cells with dihydroartemisinin at concentrations of 0, 5, 10, 20, 40, and 80 μg/ml, and with carfilzomib at concentrations of 0, 5, 10, 20, 40, and 80 nmol/L. The ARD cells were divided into a control group (no treatment) , a dihydroartemisinin group (2 μg/ml) , a carfizomib group (8 nmol/L) , and a combination group (dihydroartemisinin 2 μg/ml + carfizomib 8 nmol/L) . Cell activity and proliferation were assessed by MTT assay and EdU-488 assay; cell apoptosis was evaluated using live cell/dead cell dual staining and flow cytometry. The expression levels of apoptosis-related proteins were examined using Western blotting analysis. Results:The cell survival rates of ARD cells treated with 0, 5, 10, 20, 40, and 80 μg/ml dihydroartemisinin were (100.00±2.18) %, (50.22±3.09) %, (37.39±2.34) %, (30.42±1.79) %, (23.80±1.12) %, and (18.04±0.79) %, respectively, and there was a statistically significant difference ( F=653.30, P<0.001) . With the increase of drug concentration, ARD cell activity decreased gradually (all P<0.05) . The cell survival rates of ARD cells treated with 0, 5, 10, 20, 40, and 80 nmol/L carfilzomib were (100.00±1.12) %, (83.98±2.95) %, (67.27±2.10) %, (58.24±2.02) %, (46.34±1.14) %, and (37.47±1.36) %, respectively, and there was a statistically significant difference ( F=227.40, P<0.001) . With the increase of drug concentration, ARD cell activity decreased gradually (all P<0.05) . The cell survival rates for the control group, dihydroartemisinin group, carfilzomib group, and combination group were (100.00±2.67) %, (67.23±0.57) %, (76.23±2.83) %, and (27.06±1.09) %, respectively, and there was a statistically significant difference ( F=655.60, P<0.001) . There were statistically significant differences in the dihydroartemisinin group, carfilzomib group, and combination group compared with control group (all P<0.001) . There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group (both P<0.001) . The EdU-488 experiment showed that the EdU-positive rates of ARD cells in the control group, dihydroartemisinin group, carfilzomib group, and combination group were (100.00±8.17) %, (68.07±6.14) %, (85.04±2.78) %, and (19.62±3.83) %, respectively, and there was a statistically significant difference ( F=115.20, P<0.001) . There were statistically significant differences in the dihydroartemisinin group, carfilzomib group, and combination group compared with control group ( P<0.001; P=0.047; P<0.001) . There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group (both P<0.001) . The live cell/dead cell dual staining experiment showed, under bright-field observation, the cell morphology was intact in the control group. In all the drug groups, the cell morphology became irregular, reduced in size with condensed cytoplasmic, and apoptotic vesicles with irregular morphology were seen around the cells, among which the most obvious changes were seen in the combination group. Under fluorescence observation, the cells in the control group only displayed green fluorescence. In all drug-treated groups, cells with red fluorescence were observed, with the combination group having the highest percentage of cells with red fluorescence among the total cell population. The apoptosis rates for the control group, dihydroartemisinin group, carfilzomib group, and combination group were (9.06±2.95) %, (29.50±1.34) %, (20.77±3.00) %, and (58.23±5.13) %, respectively, and there was a statistically significant difference ( F=115.80, P<0.001) . There were statistically significant differences in the dihydroartemisinin group, carfilzomib group, and combination group compared with control group ( P<0.001; P=0.012; P<0.001) . There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group (both P<0.001) . There were statistically significant differences in the relative expression levels of P53, Cleaved-Caspase-3, Bcl-2, and Bax proteins among the control group, dihydroartemisinin group, carfilzomib group, and combination group ( F=21.76, P<0.001; F=42.87, P<0.001; F=44.27, P<0.001; F=163.50, P<0.001) . There were statistically significant differences in the dihydroartemisinin group, carfilzomib group, and combination group compared with control group (all P<0.05) . There were statistically significant differences in the dihydroartemisinin group and carfilzomib group compared with combined group (both P<0.05) . Conclusion:The combination of dihydroartemisinin and carfilzomib can synergistically inhibit the activity and proliferation of multiple myeloma ARD cells, and promote apoptosis, and the underlying mechanism may be associated with the mitochondrial apoptosis pathway.

Dihydroartemisinin promotes radiotherapy sensitivity of nasopharyngeal carcinoma CNE-2Z cells by activating chloride channels / 中国病理生理杂志

AIM:To investigate the role of ClC-3 chloride channel in the promotion of radio sensitization of na-sopharyngeal carcinoma CNE-2Z cells by dihydroartemisinin(DHA).METHODS:MTT was used to detect the inhibito-ry effect of DHA on the viability of CNE-2Z cells and normal nasopharyngeal epithelial NP69-SV40T cells,the radio sensi-tization effect of DHA on CNE-2Z cells was detected by cloning assay,the expression of ClC-3 protein was detected by Western blot,the expression of ClC-3 protein was down-regulated by siRNA technology,and the chlorine current of cells was recorded by whole cell patch-clamp technology.RESULTS:(1)Compared with NP69-SV40T cells,DHA selective-ly inhibited the proliferation of CNE-2Z cells,with IC10 values of(13.020±4.831)μmol/L and(5.244±1.050)μmol/L,respectively(P＜0.01).(2)The results of clonal formation experiments showed that DHA had a radio sensitizing effect on CNE-2Z cells,with a radio sensitization ratio of 1.9.(3)DHA could activate the chlorine channel of CNE-2Z cells and produce an outward chlorine current,but had no effect on the chlorine channel of NP69-SV40T cells.(4)DHA promoted the expression of ClC-3 chloric channel protein in CNE-2Z cells(P＜0.01).(5)Chlorine channel blocker NPPB could in-hibit the radio sensitizing effect of DHA on CNE-2Z cells by 1.84 times,and also inhibited the chlorine current activated by DHA.(6)the down-regulation of CNE-2Z ClC-3 protein could inhibit the radio sensitization effect of DHA on CNE-2Z cells by 4.19 times,and the activation of chlorine current by DHA on CNE-2Z cells was no longer produced.CONCLU-SION:DHA has a radio sensitizing effect on nasopharyngeal carcinoma CNE-2Z cells,which is likely to be related to the activation of ClC-3 chloride channel.

Effect of dihydroartemisinin on anti tumor immune response of CD8 + T cells induced by non small cell lung cancer cells / 安徽医科大学学报

Objective @#To investigate the regulatory effect of artemisinin derivative dihydroartemisinin ( DHA) on anti-tumor immune function of CD8 + T cells induced by non-small cell lung cancer ( NSCLC) cells . @*Methods@#NSCLC A549 cells were divided into DMSO control group and DHA treatment group . A549 cells were treated with DMSO and DHA at different concentrations (25 , 50 and 100 μmol/L) , and the optimal concentration of DHA was selected to treat A549 cells for 0 , 24 , 48 and 72 h according to half maximal inhibitory concentrate (IC50 ) . CCK 8 method and colony formation test were used to detect the effect of DHA on the proliferation and colony formation ability of A549 cells . Peripheral blood mononuclear cells (PBMCs ) of healthy individuals were isolated by density gradient centrifugation . After monocytes were removed by adhesion method , A549 cells pretreated with mitomycin C were co cultured with PBMCs at 10:1 ratio . After 2 weeks , flow cytometry was used to detect the proportion of CD8 + T cells and the expression levels of perforin and granzyme B .@*Results @#Compared with the control group , the proliferation inhibition rates of A549 cells increased after treatment with 25 , 50 and 100 μmol/L DHA for 24 h (P < 0.01) . The IC50 of DHA on A549 cells was 46.26 μmol/L. According to IC50 concentration analysis , the inhibi tion rates of A549 cells treated with 50 μmol/L DHA for 0 , 24 , 48 and 72h were 1 53% , 53.50% , 63.84% and 69.91% , and the cells inhibition rates of A548 cells increased compared with the previous ob servation time point , namely 0 , 24 and 48 h (P < 0.01) . The colony formation assay showed that the colony formation number of A549 cells in DHA treated group decreased compared with the control group (P < 0.01) . Flow cytometry results showed that compared with the control group , the proportion of CD8 + T cells induced by A549 cells in the co-culture system and the proportion of CD8 + T cells expressing perforin and granzyme B were higher in DHA pretreatment group(P < 0.01) . @*Conclusion @#DHA inhibits the growth of NSCLC cells and promotes anti tumor immune response of CD8 + T cells induced by NSCLC cells .

Dihydroartemisinin Suppresses Proliferation of Oral Squamous Cell Carcinoma Cells by Promoting Autophagy / 肿瘤防治研究

Objective To investigate the effect of dihydroartemisinin (DHC) on the proliferation capacity of human oral squamous carcinoma cells and its mechanism of action. Methods The viability and colony formation ability of CAL27 cells treated with different concentrations of dihydroartemisinin was measured by CCK-8 and colony formation assay. The expression of proteins related to proliferation and autophagy was determined by Western blot. Potential targets for DHA inhibition of the biological behavior of oral cancer were screened based on network pharmacology and bioinformatics. Measurement was conducted after the cells were cotreated with autophagy blocker 3-methyladenine and autophagy inducers rapamycin and dihydroartemisinin. Results Dihydroartemisinin significantly reduced the proliferation viability and clone formation ability of CAL27 cells in a concentration-dependent manner. The PCNA expression level also decreased substantially. DHA suppressed oral cancer targets involving autophagy-related pathways. DHA intervention increased the expression of intracellular autophagy-related proteins Beclin-1 and LC3. After co-treatment of DHA combined with autophagy blocker, the proliferation viability and clone formation ability of CAL27 cells decreased. The expression of PCNA increased, and the expression of Beclin-1 and LC3 decreased. Conclusion Dihydroartemisinin could inhibit the proliferative capacity of oral squamous carcinoma cells in vitro, and its effect may be correlated with the induction of autophagy.

Mechanism of dihydroartemisinin binding SERCA2b and inducing apoptosis in colorectal cell HCT-116 / 中国医科大学学报

Objective To investigate the binding sites of dihydroartemisinin(DHA)and sarcoplasmic/endoplasmic reticulum calcium ATPase(SERCA)and explore the mechanisms underlying apoptosis induction in HCT-116 colon cancer cells through the mitochondrial pathway.Methods HCT-116 cells were cultured in DHA concentrations of 0,10,20,40 μmol/L.After 24 h of culture,Western blot-ting assessed SERCA concentration,CCK-8 measured cell proliferation,Hoechst nuclear staining examined cell apoptosis,JC-1 probe evaluated mitochondrial membrane potential.LeDock molecular docking predicted DHA and SERCA binding sites.Synthetic SERCA2b mutated and non-mutated proteins at Ile315 and Thr316 sites were combined with small molecule DHA using biofilm interference tech-nology.Results Western blotting revealed a significant decrease in SERCA2 protein levels with increasing DHA concentration.CCK-8 demonstrated a statistically significant decrease in cell proliferation with increasing DHA concentration(P＜0.01).Hoechst nuclear staining illustrated DHA-induced rounding and pyknosis of HCT-116 cell nuclei compared to the control group.DHA gradually decreased mitochondrial membrane potential within the first 4 h of treatment,starting from 5 h,followed by a sustained reduction.Molecular docking predicted a hydrogen bond with Thr316 and a hydrophobic interaction with Ile315.Biofilm interference techniques indicated robust binding of DHA to non-mutated SERCA2b protein,while binding to the SERCA2b mutant protein at Ile315 and Thr316 sites was poor.Conclusion DHA directly binds to the Ile315 and Thr316 sites of SERCA2b,inducing apoptosis in colon cancer cells HCT-116 through the mitochondrial pathway.

Ginsenoside Rg_3 based liposomes target delivery of dihydroartemisinin and paclitaxel for treatment of triple-negative breast cancer / 中国中药杂志

Ginsenoside Rg_3, an active component of traditional Chinese medicine(TCM), was used as the substitute for cholesterol as the membrane material to prepare the ginsenoside Rg_3-based liposomes loaded with dihydroartemisinin and paclitaxel. The effect of the prepared drug-loading liposomes on triple-negative breast cancer in vitro was evaluated. Liposomes were prepared with the thin film hydration method, and the preparation process was optimized by single factor experiments. The physicochemical properties(e.g., particle size, Zeta potential, and stability) of the liposomes were characterized. The release behaviors of drugs in different media(pH 5.0 and pH 7.4) were evaluated. The antitumor activities of the liposomes were determined by CCK-8 on MDA-MB-231 and 4T1 cells. The cell scratch test was carried out to evaluate the effect of the liposomes on the migration of MDA-MB-231 and 4T1 cells. Further, the targeting ability of liposomes and the mechanism of lysosome escape were investigated. Finally, H9c2 cells were used to evaluate the potential cardiotoxicity of the preparation. The liposomes prepared were spheroid, with uniform particle size distribution, the ave-rage particle size of(107.81±0.01) nm, and the Zeta potential of(2.78±0.66) mV. The encapsulation efficiency of dihydroartemisinin and paclitaxel was 57.76%±1.38% and 99.66%±0.07%, respectively, and the total drug loading was 4.46%±0.71%. The accumulated release of dihydroartemisinin and paclitaxel from the liposomes at pH 5.0 was better than that at pH 7.4, and the liposomes could be stored at low temperature for seven days with good stability. Twenty-four hours after administration, the inhibition rates of the ginsenoside Rg_3-based liposomes loaded with dihydroartemisinin(70 μmol·L~(-1)) and paclitaxel on MDA-MB-231 and 4T1 cells were higher than those of the positive control(adriamycin) and free drugs(P<0.01). Compared with free drugs, liposomes inhibited the migration of MDA-MB-231 and 4T1 cells(P<0.05). Liposomes demonstrated active targeting and lysosome escape. In particular, liposomes showed lower toxicity to H9c2 cells than free drugs(P<0.05), which indicated that the preparation had the potential to reduce cardiotoxicity. The findings prove that ginsenoside Rg_3 characterized by the combination of drug and excipient is an ideal substitute for lipids in liposomes and promoted the development of innovative TCM drugs for treating cancer.

Synergistic mechanisms of ferroptosis in anaplastic thyroid cancer induced by dihydroartemisinin and sorafenib / 中华内分泌代谢杂志

Objective:To investigate the synergistic effects and molecular mechanisms of dihydroartemisinin(DHA) and sorafenib(SOR) in inducing ferroptosis in anaplastic thyroid cancer(ATC) cells.Methods:CCK-8 and flow cytometry assays were performed to detect the effects of DHA and SOR on the proliferation and ferroptosis of ATC cells(CAL-62). Real-time fluorescence quantitative PCR and Western blotting assays were performed to detect the expressions of ferroptosis-related genes glutathione peroxidase 4(GPX4), solute carrier family 7 member 11 gene(SCL7A11), lipoxygenase-15(LOX-15), and p53. The levels of iron death intermediate metabolites including lactate dehydrogenase(LDH), glutathione(GSH), malondialdehyde(MDA), ferrous ion(Fe 2+ ), nitric oxide(NO), and reactive oxygen species(ROS)were measured by corresponding assay kits. The corresponding inhibition of DHA and SOR on ATC in vivo was analyzed in a tumor model in nude mice. Results:Compared with the control group, DHA, SOR, and DHA+ SOR treatment significantly inhibited cell proliferation and apoptosis in a dose-dependent manner( P<0.001), with increased LDH, Fe 2+, MDA, and ROS contents and reduced GSH activity( P<0.001), which were promoted by ferrous sulfate(FeSO 4)and reversed by ferroptosis inhibitor-1. Compared with the control group and the drug monotherapy group, 15-LOX-2 and p53 expressions were upregulated in DHA+ SOR group while GPX4 and SCL7A11 expressions were decreased( P<0.001), without significant difference in 15-LOX-1 protein content. In addition, NO level was significantly increased in DHA+ SOR group( P<0.001). DHA and SOR inhibited tumor growth of ATC in vivo. Conclusion:DHA and SOR synergistically induced ferroptosis via upregulating the expression of 15-LOX-2 gene and inhibiting NO synthesis in ATC cells.

Dihydroartemisinin ameliorates innate inflammatory response induced by Streptococcus suis-derived muramidase-released protein via inactivation of TLR4-dependent NF-κB signaling / 药物分析学报

Muramidase-released protein(MRP)is now being recognized as a critical indicator of the virulence and pathogenicity of Streptococcus suis(S.suis).However,the identification of viable therapeutics for S.suis infection was hindered by the absence of an explicit mechanism for MRP-actuated inflammation.Dihydroartemisinin(DhA)is an artemisinin derivative with potential anti-inflammatory activity.The modulatory effect of DhA on the inflammatory response mediated by the virulence factor MRP remains obscure.This research aimed to identify the signaling mechanism by which MRP triggers the innate immune response in mouse spleen and cultured macrophages.With the candidate mechanism in mind,we investigated DhA for its ability to dampen the pro-inflammatory response induced by MRP.The innate immune response in mice was drastically triggered by MRP,manifesting as splenic and systemic inflammation with splenomegaly,immune cell infiltration,and an elevation in pro-inflammatory cyto-kines.A crucial role for Toll-like receptor 4(TLR4)in coordinating the MRP-mediated inflammatory response via nuclear factor-kappa B(NF-κB)activation was revealed by TLR4 blockade.In addition,NF-KB-dependent transducer and activator of transcription 3(STAT3)and mitogen-activated protein kinases(MAPKs)activation was required for the inflammatory signal transduction engendered by MRP.Intriguingly,we observed an alleviation effect of DhA on the MRP-induced immune response,which referred to the suppression of TLR4-mediated actuation of NF-KB-STAT3/MAPK cascades.The inflam-matory response elicited by MRP is relevant toTLR4-dependent NF-κB activation,followed by an increase in the activity of STAT3 or MAPKs.DhA mitigates the inflammation process induced by MRP via blocking the TLR4 cascade,highlighting the therapeutic potential of DhA in targeting S.suis infection diseases.

Dihydroartemisinin Regulates Neuro-microglia to Relieve Neuropathic Pain / 中国实验方剂学杂志

ObjectiveTo evaluate the intervention effect of dihydroartemisinin （DHA） on hippocampal nerve injury in L5 spinal nerve ligation （SNL） model and tumor necrosis factor-α （TNF-α） hippocampal continuous injection model. In primary cultured microglia-hippocampal neurons， the regulatory pattern of DHA on microglia-hippocampal neuronal interactions was confirmed. MethodThe experimental animals were divided into Sham group， SNL group， and DHA group （16 mg·kg-1）， with 3 mice in each group. The hippocampal CA3 glutamatergic neurons were labeled with adeno-associated virus ［Calmodulin-dependent protein kinase Ⅱ（CaMKⅡ） dTomato AAV］， and their contributions to the hippocampal CA1， prefrontal cortex （Frc）， anterior cortex （ACC）， projections of nucleus accumbens （Nac）， and Basolateral Amygdala （BLA） were traced by immunofluorescence staining. The experimental animals were divided into a Sham group， a TNF-α hippocampus continuous injection model group， DHA-L， DHA-M， and DHA-H groups （4， 8， 16 mg·kg-1）， and pregabalin group （25 mg·kg-1）， with 4 mice in each group. The morphology of pyramidal neurons in the hippocampal CA1 and CA3 regions was counted by Golgi staining. The continuous activation of hippocampal primary neurons and microglia was induced， DHA intervention was given by co-culture， and the cell soma area and the expression of postsynaptic density protein 95 （PSD95） inside and outside the primary and secondary dendritic spines of neurons were counted by immunofluorescence. ResultCompared with the Sham group， the projection of CA3 glutamatergic neurons to CA1 region， Frc， and ACC in the SNL group was significantly reduced （P<0.01）， while the projection to Nac and BLA was significantly increased （P<0.01）. As compared with the SNL group， the projection of hippocampal CA3 glutamatergic neurons to CA1 region， Frc， and ACC was significantly increased in the DHA group （P<0.01）， while the projection to Nac and BLA was significantly reduced （P<0.01）. Golgi staining results showed that as compared with the Sham group， the density of dendritic spines and the number of dendritic branches in the CA1 and CA3 pyramidal neurons in the TNF-α hippocampal continuous injection model group were significantly reduced （P<0.01）. As compared with the TNF-α hippocampal continuous injection model， the density of dendritic spines and the number of dendritic branches in hippocampal CA1 and CA3 pyramidal neurons in the DHA-M and DHA-H groups were significantly increased （P<0.05， P<0.01）. Compared with DHA-M group， the total dendrite length of CA1 pyramidal neurons in hippocampus in DHA-H group was significantly increased （P<0.01）， while the total dendrite length of CA1 neurons and the total dendrite base length of CA3 neurons in DHA-L group was significantly decreased （P<0.01）. Compared with the blank control group， the cell soma area of the glycine group and glutamate group increased significantly （P<0.01）. As compared with the glycine group and glutamate group， the cell area of the glycine + glutamate group was significantly increased （P<0.01）， and as compared with the glutamate group， the cell soma area of the glutamate + DHA group was significantly reduced （P<0.01）. As compared with the glycine acid + glutamate group， the cell soma area of the glycine + glutamate + DHA group was significantly reduced （P<0.01）， and as compared with the glutamate + DHA group， the cell soma area of the glycine + glutamate + DHA group was also significantly reduced （P<0.05）. Compared with the blank control group， the cell soma area of the glutamate group was significantly increased （P<0.01）. As compared with the glutamate group， the cell soma area of the glutamate + DHA-L， glutamate + DHA-M， and glutamate + DHA-H groups was significantly reduced （P<0.01）. As compared with the blank control group， the expression of the resting primary microglia + glycine group in primary and secondary dendritic internal and external postsynaptic density protein 95 （PSD95） was significantly increased （P<0.01）. As compared with the resting primary microglia + glycine group， the expression of PSD95 in the primary and secondary dendritic spinous and external neurons of the activated primary microglia + glycine group was significantly reduced （P<0.01）. As compared with the activated primary microglia + glycine group， the expression of PSD95 in the primary and secondary dendritic spinous and external neurons in the activated primary microglia + glycine + DHA group was significantly increased （P<0.01）. As compared with the activated primary microglia + DHA group， the expression of PSD95 in the primary and secondary dendritic spines and outside neurons in the activated primary microglia + glycine + DHA group was significantly increased （P<0.01）. ConclusionDHA has a significant repair effect on vertebral neuronal damage caused by hippocampal microglia and TNF-α overexpression in NP pathology， and this repair is closely related to the dual inhibition of neuronal-microglia by DHA.

Dihydroartemisinin increased the abundance of Akkermansia muciniphila by YAP1 depression that sensitizes hepatocellular carcinoma to anti-PD-1 immunotherapy / 医学前沿

The effect of anti-programmed cell death 1 (anti-PD-1) immunotherapy is limited in patients with hepatocellular carcinoma (HCC). Yes-associated protein 1 (YAP1) expression increased in liver tumor cells in early HCC, and Akkermansia muciniphila abundance decreased in the colon. The response to anti-PD-1 treatment is associated with A. muciniphila abundance in many tumors. However, the interaction between A. muciniphila abundance and YAP1 expression remains unclear in HCC. Here, anti-PD-1 treatment decreased A. muciniphila abundance in the colon, but increased YAP1 expression in the tumor cells by mice with liver tumors in situ. Mechanistically, hepatocyte-specific Yap1 knockout (Yap1LKO) maintained bile acid homeostasis in the liver, resulting in an increased abundance of A. muciniphila in the colon. Yap1 knockout enhanced anti-PD-1 efficacy. Therefore, YAP1 inhibition is a potential target for increasing A. muciniphila abundance to promote anti-PD-1 efficacy in liver tumors. Dihydroartemisinin (DHA), acting as YAP1 inhibitor, increased A. muciniphila abundance to sensitize anti-PD-1 therapy. A. muciniphila by gavage increased the number and activation of CD8+ T cells in liver tumor niches during DHA treatment or combination with anti-PD-1. Our findings suggested that the combination anti-PD-1 with DHA is an effective strategy for liver tumor treatment.

Dihydroartemisinin attenuates ischemia/reperfusion-induced renal tubular senescence by activating autophagy / 中国天然药物

Acute kidney injury (AKI) is an important factor for the occurrence and development of CKD. The protective effect of dihydroartemisinin on AKI and and reported mechanism have not been reported. In this study, we used two animal models including ischemia-reperfusion and UUO, as well as a high-glucose-stimulated HK-2 cell model, to evaluate the protective effect of dihydroartemisinin on premature senescence of renal tubular epithelial cells in vitro and in vivo. We demonstrated that dihydroartemisinin improved renal aging and renal injury by activating autophagy. In addition, we found that co-treatment with chloroquine, an autophagy inhibitor, abolished the anti-renal aging effect of dihydroartemisinin in vitro. These findings suggested that activation of autophagy/elimination of senescent cell might be a useful strategy to prevent AKI/UUO induced renal tubular senescence and fibrosis.

Mechanisms of amelioration of lipopolysaccharide-induced acute lung injury in mice by dihydroartemisinin using whole-genome sequencing / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect and mechanism of dihydroartemisinin(DHA)on lipo-polysaccharide(LPS)-induced acute lung injury(ALI)in mice using whole-genome sequencing.METHODS An ALI mouse model was established via intraperitoneal injection of 10 mg·kg-1 lipopolysaccharide.The mice were divided into normal control group(n=10),model group(n=10)and model+DHA group(n=10).The mice in the model+DHA group were injected intraperitoneally with 20 mg·kg-1 DHA,while those in the normal control group and LPS group were injected intraperitoneally with solvent of DHA,saline containing 1%Tween 80 and 10%Macrogol 400.The mice were executed 24 h after drug administration.The wet and dry weight ratio(W/D)of lung tissue was calculated.Hematoxylin-eosin(HE)staining was used to observe histopathological damage in the lung.Classified counts of inflamma-tory cells in alveolar lavage fluid were performed.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-1β(IL-1β),IL-6,and tumor necrosis factor-α(TNF-α)in alveolar lavage fluid.Real-time quantitative PCR(RT-qPCR)was used to detect mRNA levels of placenta-specific 8(Plac8),Toll-like receptor 7(TLR7),IL-1β,IL-6 and TNF-αin lung tissue.The whole gene transcriptome was sequenced by RNA transcriptome sequencing(RNA-seq)using the Illumina HiSeq high-throughput sequencing platform before the function and signal pathway of differentially expressed gene mRNA between the groups were enriched and analyzed using GO and KEGG enrichment analysis methods.RESULTS Compared with the model group,the lung W/D values of mice,the pathological damage,inflammatory cells in alveolar lavage fluid,expression levels of IL-1β,IL-6 and TNF-α in alveolar lavage fluid(P<0.01,P<0.01,P<0.01),and the mRNA expression levels of IL-1β,IL-6 and TNF-α were significantly reduced in lung tissues in the model+DHA group(P<0.01,P<0.05,P<0.05).Whole gene transcriptome sequencing revealed that immune-related Plac8 and TLR7 genes were significantly upregu-lated(P<0.01)in mouse lung tissue of the model group but significantly downregulated(P<0.05)in mouse lung tissue of the model+DHA group.The results of RT-qPCR of Plac8 and TLR7 verified the results of whole gene transcriptome sequencing.GO and KEGG analysis showed that Plac8 and TLR7 were mainly related to the regulation of cytokine production,T/B cell activation and signal transduction,chemo-kine signal transduction and NF-κB signal transduction.CONCLUSION DHA might reduce LPS-induced lung damage and ameliorate the inflammatory condition in lungs of ALI mice.The mechanism of action may be that DHA negatively regulates the signaling pathways involved in TLR7 and Plac8 by decreasing the expressions of TLR7 and Plac8 mRNA before regulating a series of immune responses such as secretion of inflammation-related cytokines and activation of immune cells,thereby reducing inflam-matory damage in lungs.

Characteristics and artemisinin based treatment effect of 362 malaria cases in the peacekeeping mission area of the Democratic Republic of the Congo / 中华地方病学杂志

Objective:To analyze the epidemiological characteristics, diagnosis and treatment of malaria in the peacekeeping mission area of the Democratic Republic of the Congo (DRC), and to evaluate the efficacy of artemisinin based combination therapy (ACT), to provide clinical basis for the diagnosis and treatment of malaria.Methods:The clinical data of malaria-infected peacekeepers admitted to the Chinese Secondary Hospital of the United Nations Stabilization Mission in DRC (MONUSCO) from January 2014 to September 2020 were collected, and the general information, incidence characteristics, treatment and clinical outcomes of the patients were retrospectively analyzed.Results:From January 2014 to September 2020, 362 peacekeepers were hospitalized with malaria, the average annual incidence case was 54 cases per year, and the annual incidence was 9.5/1 000, with a median of 2.5 days (1 - 9 days) from onset to diagnosis. Severe malaria accounted for 7.73% (28/362) and uncomplicated malaria accounted for 92.27% (334/362). The incidence rate was 37.57% (136/362) in the dry season (April to September) and 62.43% (226/362) in the rainy season (October to March of the following year). After ACT antimalarial treatment, all patients were cured clinically. Eight cases recurred and were cured clinically after drug conversion ACT retreatment.Conclusions:In the peacekeeping mission area of DRC, peacekeepers are generally susceptible to malaria. ACT has a high cure rate, safety and efficiency in clinical treatment.

Stability，in vitro Release and Tissue Distribution of Docetaxel-dihydroartemisinin Conjugated Prodrug Self- assembled Nanoparticles / 中国药房

OBJECTIVE：To study the sta bility，in vivo release characteristics and tissue distribution of docetaxel （DTX）- dihydroartemisinin（DHA）conjugated prodrug self-assembled nanoparticles （DTX-S-S-DHA NPs ）. METHODS ：HPLC method was adopted to analyze DTX-S-S-DHA in vitro . The phycial and long-term stability of DTX-S-S-DHA NPs in mediums [water ， saline，phosphate buffer （PBS，pH 7.4）and RPMI 1640 medium] were investigated by using particle size ，polydispersity index （PDI）and encapsulation efficiency （EE）as evaluation indexes. The in vitro release characteristics of DTX-S-S-DHA released from DTX-S-S-DHA NPs was also investigated with small glass method ，using 30％ ethanol solution with or without 10 mmol/L dithiothreitol（DTT）as medium. The small live animal imager was adopted to investigate the tissue distribution and tumor targeting capability of DiR-labeled DTX-S-S-DHA NPs （DTX-S-S-DHA/DiR NPs ）in breast cancer bearing mice. RESULTS ：In stability test，there was no statistical difference in particle size ，PDI and EE of DTX-S-S-DHA NPs incubated in water ，normal saline ，PBS and RPMI 1640 medium for 24 h. When stored at 4 ℃，with the increase of storage time ，the particle size of DTX-S-S-DHA NPs in normal saline gradually increased ，while those in PBS gradually decreased ；EE of both gradually decreased to less than 75％， but there was no significant change in particle size ，PDI and EE of DTX-S-S-DHA NPs in water and RPMI 1640 medium. In the in vitro release experiments ，DTX-S-S-DHA in DTX-S-S-DHA NPs was not released in the release medium containing 10 mmol/L DTT；at 24 h，the cumulative release rate of DTX-S-S-DHA released from DTX-S-S-DHA NPs in release medium without DTT was about 83％，which was in line with first-order kinetic model. In tissue distribution test ，the distribution of DTX-S-S-DHA/DiR NPs in tumor sites of mice was significantly more than in other tissues （heart，liver，spleen，lung and kidney ）. CONCLUSIONS ： DTX-S-S-DHA NPs show good physical stability in different mediums ，especially have good long-term stability in water and RPMI ； 1640 medium；they can quickly release the parent drug in the reduction environment and has good tumor targeting.

Effect of Dihydroartemisinin on Oxidative Damage and Energy Metabolism of HepG2 Cells and Its Synergistic Effect with Sorafenib / 中国实验方剂学杂志

Objective:To explore the inhibitory effect of dihydroartemisinin （DHA） on the proliferation of HepG2 cells， elucidate the mechanism from the perspectives of oxidative damage and energy metabolism， and discuss the possibility of combined use of DHA with sorafenib （Sora）. Method:Cell counting kit-8 （CCK-8） assay was used to obtain the 50% inhibitory concentration （IC₅₀） of DHA and Sora on HepG2 and SW480 cells and Chou-Talalay method was used to obtain the combination index （CI） of DHA and Sora. HepG2 cells were classified into the control group， DHA group （10 µmol·L^-1）， Sora group （5 µmol·L^-1）， and DHA + Sora group （DHA 10 µmol·L^-1， Sora 5 µmol·L^-1） and then incubated with corresponding drugs for 8-12 h. Seahorse XF glycolytic rate assay kit and cell mito stress test kit were employed to respectively detect the glycolysis function of cells and oxidative phosphorylation function of mitochondria. DCFH-DA and lipid peroxidation MDA assay kit were separately used to analyze the intracellular levels of reactive oxygen species （ROS） and malondialdehyde （MDA）. Western blot was applied to determine the intracellular levels of heme oxygenase-1 （HO-1） and glutamate-cysteine ligase catalytic subunit （GCLC）. Result:Compared with the control group， DHA alone inhibited the ATP synthesis in mitochondrial oxidative phosphorylation and glycolysis （<italic>P</italic><0.01）， increased the levels of intracellular ROS and MDA （<italic>P<</italic>0.05）， and decreased the levels of HO-1 and GCLC （<italic>P<</italic>0.05） in HepG2 cells. DHA and Sora had synergistic inhibitory effect on proliferation of HepG2 and SW480 cells， with CI < 0.90. The DHA + Sora group showed stronger suppression of ATP synthesis in mitochondrial oxidative phosphorylation and glycolysis （<italic>P</italic><0.01）， higher levels of intracellular ROS and MDA （<italic>P<</italic>0.01）， and lower levels of intracellular antioxidation-related proteins HO-1 and GCLC in HepG2 cells （<italic>P<</italic>0.01） than the DHA group. Conclusion:DHA may increase the level of MDA by reducing HO-1 and GCLC and increasing ROS in HepG2 cells， which results in mitochondria oxidative damage， restricts cell glycolysis and mitochondrial oxidative phosphorylation， and thus finally inhibits the proliferation of HepG2 cells. DHA and Sora have synergistic inhibitory effect on the proliferation of HepG2 and SW480 cells， and the mechanism may be related to the synergistic oxidative damage that affects the mitochondrial electron transport chain and suppresses cell energy metabolism.

Dihydroartemisinin and its anticancer activity against endometrial carcinoma and cervical cancer: involvement of apoptosis, autophagy and transferrin receptor

INTRODUCTION@#Dihydroartemisinin (DHA) is a first-line antimalarial drug with relatively low toxicity. DHA has been speculated to possess a broad-spectrum antitumour effect. However, the potential value of DHA for the treatment of endometrial carcinoma or cervical cancer is unclear.@*METHODS@#We used human endometrial cancer cells and cervical cancer cells to assess whether DHA alone or when combined with cisplatin would induce cell death. We aimed to elucidate the role of autophagy in DHA-induced cytotoxicity in both endometrial and cervical cancer cells, and explore the impact of DHA treatment on cell proliferation, apoptosis and autophagy.@*RESULTS@#DHA alone or in combination with cisplatin induced cell death in a dose- and time-dependent manner. Caspase-3 mRNA and cleaved caspase-3 protein levels were markedly elevated following DHA treatment either in the presence or absence of cisplatin, suggesting a role of apoptosis in DHA-induced cell death. DHA treatment activated the autophagic pathway, as evidenced by increased monodansylcadaverine-positive staining, elevated microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I ratio, and enhanced p62/sequestosome 1 degradation. Inhibition of autophagy by 3-methyladenine further enhanced the cytotoxicity of DHA towards tumour cells. mRNA levels of transferrin receptor (TfR) were suppressed upon DHA treatment and knockdown of TfR by RNA interference caused further DHA induction of cancer cell death.@*CONCLUSION@#Our results suggest a clinical value for DHA in the treatment of endometrial carcinoma and cervical cancer. Our data revealed possible anticancer mechanisms of DHA that involve regulating apoptosis, autophagy pathway and levels of TfR.

Dihydroartemisinin-piperaquine for the routine treatment of uncomplicated malaria in Northern Ghana

Background:Dihydroartemisinin-piperaquine is a first line treatment for uncomplicated malaria in Ghana. A facility-based study was undertaken to examine the effectiveness of thetreatment in the routine health care system.Methods:The study was undertaken at the Navrongodemographic surveillance area. Patients presenting with acute febrile illness were enrolled after informed consented and confirmation by microscopy. Patients were randomized into supervised group who received treatment under direct observation and unsupervised group which had only the first treatment given under supervision. Treatment was according to bodyweight and 42 days follow-up was undertaken.Results:A total of 194 patients were enrolled; 54.1% were females and 51% had supervised treatment. The median age and weight were 6.7 years and 20.0kg respectively. Mean baseline temperature, haemoglobin concentration and parasite density were, 37.6oC, 11.1 g/dl and 11,098 parasites per microliter of blood respectively. Study completion rate was 93.3%, day 42 polymerase chain reaction-unadjusted adequate clinical and parasitological responses rate (ACPR) was 93.4% by evaluable and 87.1 % by intention-to-treat (ITT). The day 42 ACPR by evaluable was 92.3% in the supervised arm compared to 94.4% in the unsupervised arm. The day 42 ACPR by ITT was 85.7% in the supervised and 88.5% in the unsupervised arms. The fever resolution and haemoglobin concentration changes for the two arms were similar.Conclusions: The results show that dihydroartemisinin-piperaquine iseffective and good first-line antimalarial in the routine health delivery system

Effect of Arsenic Trioxide Combined with Dihydroartemisinin on THP-1 Cells Proliferation and Apoptosis / 中国实验方剂学杂志

Objective:To investigate the effects of arsenic trioxide combined with dihydroartemisinin on proliferation, cell cycle, and apoptosis of THP-1 cells, and explore the mechanism. Method:The thiazolyl blue （MTT） method was applied to detect the effect of different concentrations of arsenic trioxide, dihydroartemisinin and arsenic trioxide combined with dihydroartemisinin on the proliferation of THP-1 cells. Annexin V/propidium iodide（PI）assay was used to detect the change of THP-1 cell cycle and apoptosis．Western blot was performed to assess the expression of cysteine protease-3（Caspase-3）, cleaved Caspase-3, B-lymphocytoma-2（Bcl-2） and Bcl-2 associated X protein （Bax）. The changes of cell morphology were observed under high intension microscope. Result:Compared with blank group, arsenic trioxide and dihydroartemisinin both exhibited obvious antiproliferative effect on the human acute monocytic leukemia cell line THP-1 in time-dose dependence （P<0.01）. After 48 h, compared with the same dose of arsenic trioxide or that of dihydroartemisinin alone, the inhibition effect of 1 µmol·L-1 arsenic trioxide combined with 2 µmol·L-1 dihydroartemisinin on proliferation of THP-1 cells was significantly stronger （P<0.01）. Compared with the control group, arsenic trioxide combined with dihydroartemisinin significantly arrested the cells in G1 phase （P<0.01）, induced the downregulation of Caspase-3 and Bcl-2 （P<0.01） and upregulation of cleaved Caspase-3 significantly（P<0.05）. Conclusion:Arsenic trioxide combined with dihydroartemisinin can significantly inhibit the proliferation and induce apoptosis of THP-1 cells. The possible mechanism may be related to arrest the cells in G1 phase, reduce the expression of Caspase-3 and Bcl-2, increase the expression of cleaved Caspase-3.

Determination of Diketo Aldehyde in Dihydroartemisinin Bulk Drug by Principal Component Reference Substances External Standard Method with Correction Factor / 中国药学杂志

OBJECTIVE: To establish a principal component reference substances external standard method with correction factor for the determination of impurity diketoal dehyde (DKA) in diketo aldehyde(DHA) bulk drug by selecting suitable liquid phase conditions. METHODS: The chromatographic C18 column (4.6 mm×250 mm, 5 μm) was used; isometric elution was set at conducted using acetonitrile-water (37∶63) as elution condition; the flow rate was 1 mL•min-1; the detection wavelength was 216 nm; the column temperature was maintained at 15 ℃; the injection volume was 20 μL. RESULTS: The DHA α peak and its impurity DKA were well separated. The average correction factor was 0.256 determined by three different chromatographic columns. CONCLUSION: The correction factor of DKA in DHA is accurate and reliable. This method can be used for the quality control of DKA in DHA raw materials.

Study on the Effect and Its Mechanism of Dihydroartemisinin on the Metabolism of Amino Acids in Hepatoma Cells / 中国药房

OBJECTIVE：To investigate the effects of dihydroartemisinin （DHA）on the metabolism of amino acid metabolites in human hepatocellular carcinoma cells Huh 7 and BEL- 7402，and to provide theoretic basis for clarifying the mechanism of DHA regulating the metabolism of hepatocellular carcinoma cells. METHODS ：CCK-8 method was taken to detect the effect of different concentrations of DHA （12.5，25，50，100 µmol/L）treating for 24，48，72 h on the two kinds of cells. Two kinds of cells were divided into control group and administration group （DHA，25 µmol/L），and then cultured with drug-free or drug-containing medium for 24 h，operated in parallel for three times. After derivatization of cell samples in each group ，GC-MS method was used to detect the content of amino acid metabolites ，combined with SIMCA-P software analysis and compound library comparison ，the differential metabolites in two kinds of cells were screened out. The pathway enrichment analysis of differential metabolism was conducted with Metaboanalyst 4.0 software. RESULTS ：Compared with control group ，the contents of glutamine ，glutathione， phenylalanine，fumaric acid and taurine were trending down in Huh 7 or BEL- 7402 cells. There were 28 and 29 differential metabolites obtained from the above two kinds of cells ，and 10 of them were common differential metabolites ，including glutamine，glutathione，taurine，fumaric acid ，phenylalanine，etc. The differential metabolites were enriched in 8 and 6 pathways respectively. The common enrichment pathways were amino acid-tRNA biosynthesis ，aspartate-alanine-glutamate metabolism ， nitrogen metabolism ，phenylalanine metabolism and pentose phosphate pathway ，etc. CONCLUSIONS ：DHA can significantly reduce the activities of Huh 7 cells and BEL- 7402 cells，and the contents of glutamine ，glutamic acid ，glutathione and phenylalanine，etc. It may regulate the growth of the two kinds of cells by influencing the mechanism of aspartic acid- alanine-glutamate metabolic pathway ，etc.