Inter-Species Pharmacokinetic Modeling and Scaling for Drug Repurposing of Pyronaridine and Artesunate.

Even though several new targets (mostly viral infection) for drug repurposing of pyronaridine and artesunate have recently emerged in vitro and in vivo, inter-species pharmacokinetic (PK) data that can extend nonclinical efficacy to humans has not been reported over 30 years of usage. Since extrapolation of animal PK data to those of humans is essential to predict clinical outcomes for drug repurposing, this study aimed to investigate inter-species PK differences in three animal species (hamster, rat, and dog) and to support clinical translation of a fixed-dose combination of pyronaridine and artesunate. PK parameters (e.g., steady-state volume of distribution (Vss), clearance (CL), area under the concentration-time curve (AUC), mean residence time (MRT), etc.) of pyronaridine, artesunate, and dihydroartemisinin (an active metabolite of artesunate) were determined by non-compartmental analysis. In addition, one- or two-compartment PK modeling was performed to support inter-species scaling. The PK models appropriately described the blood concentrations of pyronaridine, artesunate, and dihydroartemisinin in all animal species, and the estimated PK parameters in three species were integrated for inter-species allometric scaling to predict human PKs. The simple allometric equation (Y = a × Wb) well explained the relationship between PK parameters and the actual body weight of animal species. The results from the study could be used as a basis for drug repurposing and support determining the effective dosage regimen for new indications based on in vitro/in vivo efficacy data and predicted human PKs in initial clinical trials.

Based on NF-κB and Notch1/Hes1 Signaling Pathways, the Mechanism of Artesunate on Inflammation in Osteoporosis in Ovariectomized Rats was Investigated.

BACKGROUND: Artesunate (ART) has the potential to modulate the nuclear factor kappa B (NF-κB) and Notch1/Hes1 signaling pathways, which play crucial roles in the pathogenesis of osteoporosis. This study aims to explore whether ART participates in the progression of osteoporosis by regulating these signaling pathways. METHODS: In the in vitro experiments, we treated bone marrow mesenchymal stem cells (BMSCs) with different concentrations of ART (0, 3, 6, 12 µM) and evaluated osteogenic differentiation using alkaline phosphatase staining (ALP) and alizarin red S staining (ARS) staining. The expression levels of osteocalcin (OCN), RUNT-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), and receptor activator of the nuclear factor kappa ligand (RANKL) were detected by real-time quantitative PCR (RT-qPCR). The effects of ART on NF-κB p65 and Notch1 protein expression were analyzed by Western blot (WB) and immunofluorescence (IF). In the in vivo experiments, a postmenopausal osteoporosis rat model was established via ovariectomy. Bone tissue pathological injury was evaluated using hematoxylin eosin (HE) staining. Serum ALP levels were measured using a kit, bone density was determined by dual-energy X-ray absorptiometry, and serum levels of bone gla protein (BGP), OPG, RANKL, tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and IL-1ß were measured by enzyme-linked immunosorbent assay (ELISA). Additionally, the expression of NF-κB p65 and Notch1 in tissues was assessed by immunohistochemistry. RESULTS: In vitro experiments revealed that compared to the control group, ART dose-dependently promoted BMSCs proliferation and enhanced their osteogenic differentiation capability. The expression of OCN, RUNX2, and OPG significantly increased in the ART-treated group, while RANKL expression decreased significantly (p < 0.05). ART significantly inhibited the expression of NF-κB p65 and Notch1/Hes1 signaling pathway proteins (p < 0.05). Compared to ART treatment alone, combined treatment with ART and phorbol myristate acetate (PMA) or valproic acid (VPA) resulted in increased expression of NF-κB p65 and Notch1 proteins and decreased osteogenic differentiation capability (p < 0.05). In vivo experiments showed that in rats treated with ART, bone damage was significantly reduced, bone density and mineral content were restored considerably, and the expression of inflammatory factors (TNF-α, IL-6, IL-1ß) decreased significantly (p < 0.05). Additionally, ART treatment significantly reduced the expression of NF-κB p65 and Notch1 proteins, increased OPG expression, and decreased BGP and RANKL levels (p < 0.05). CONCLUSION: In summary, ART facilitates the osteogenic differentiation of BMSCs by inhibiting the NF-κB and Notch1/Hes1 signaling pathways, thereby exerting significant protective effects against osteoporosis.

Artesunate induces melanoma cell ferroptosis and augments antitumor immunity through targeting Ido1.

Artesunate (ART), a natural product isolated from traditional Chinese plant Artemisia annua, has not been extensively explored for its anti-melanoma properties. In our study, we found that ART inhibited melanoma cell proliferation and induced melanoma cell ferroptosis. Mechanistic study revealed that ART directly targets Ido1, thereby suppressing Hic1-mediated transcription suppression of Hmox1, resulting in melanoma cell ferroptosis. In CD8+ T cells, ART does not cause cell ferroptosis due to the low expression of Hmox1. It also targets Ido1, elevating tryptophan levels, which inhibits NFATc1-mediated PD1 transcription, consequently activating CD8+ T cells. Our study uncovered a potent and synergistic anti-melanoma efficacy arising from ART-induced melanoma cell ferroptosis and concurrently enhancing CD8+ T cell-mediated immune response both in vivo and in vitro through directly targeting Ido1. Our study provides a novel mechanistic basis for the utilization of ART as an Ido1 inhibitor and application in clinical melanoma treatment.

Plasmodium falciparum neonatal malaria with atypical presentation: a case series from southwestern Ethiopia.

BACKGROUND: Neonatal malaria is defined as the detection of asexual stages of Plasmodium species in the cord blood within the first 28 days of life. It can be congenital or acquired through mosquito bites or blood transfusions. Neonates are generally considered to be relatively protected due to the multiple innate and acquired physiological protective effects present in neonates. However, in areas where malaria is endemic, the prevalence of malaria in neonates is high. The predominant clinical feature of malaria in neonates is fever. Other clinical manifestations of neonatal malaria include respiratory distress, pallor and anaemia, hepatomegaly, refusal to feed, jaundice and diarrhoea. Atypical presentations without fever can lead to inaccurate diagnosis and contribute to neonatal morbidity and mortality. Neonates from endemic areas with any of the above symptoms should be screened for malaria. CASE PRESENTATION: We present a series of three cases of neonatal Plasmodium falciparum malaria that presented atypically without febrile episodes and were diagnosed and managed at Mizan-Tepi University Teaching Hospital between July and September 2023. The first patient presented with vomiting, refusal to feed, pallor, severe anaemia, and splenomegaly. The second patient presented with an inconsolable cry, failure to pass feces, abdominal distention, and anaemia. The third patient presented with vomiting and anaemia. All patients received a 7-day course of intravenous artesunate; the first patient also received a blood transfusion. All patients recovered and were discharged. CONCLUSIONS: Partial immunity resulting from repeated malaria infections in endemic regions may result in the transfer of high levels of maternal Immunoglobulin G (IgG) antibodies through the placenta and can produce different atypical clinical presentations. In malaria-endemic areas, neonates presenting with any of the presenting signs and symptoms of malaria, including afebrile presentation, require malaria screening to avoid delays in diagnosis.

Exchange transfusion combined with artesunate (ET-AS) as a safe and effective therapy in severe P. falciparum malaria: a case series.

BACKGROUND: the mortality associated with severe malaria due to Plasmodiun falciparum remains high despite improvements in malaria management. Case prensentation: this case series aims to describe the efficacy and safety of the exchange transfusion combined with artesunate (ET-AS) regimen in severe P. falciparum malaria. Eight patients diagnosed with severe P. falciparum malaria were included. All patients underwent ET using the COBE Spectra system. The aimed for a post-exchange hematocrit of 30%. Half the estimated blood volume was removed and replaced using fresh frozen plasma. The regimen was well-tolerated without complications. The parasite clearance time ranged from 1 ~ 5 days. Five patients with cerebral malaria exhibited full improved consciousness within 3 days, while patient2 with hemolysis improved on day 2. Liver function improved within 1 ~ 6 days, and patient 1 and patient 6 showed improvements renal function on days 18 and 19, respectively. The length of intensive care unit stay range from 2 ~ 10 days, and all patients treated with ET-AS remained in the hospital for 3 ~ 19 days. CONCLUSIONS: these preliminary results suggest that ET-AS regimens are a safe and effective therapy for severe P. falciparum malaria and can benefit patients in clinical settings.

The human malaria-Aotus monkey model: a historical perspective in antimalarial chemotherapy research at the Gorgas Memorial Laboratory-Panama.

The human malaria-Aotus monkey model has served the malaria research community since its inception in 1966 at the Gorgas Memorial Laboratory (GML) in Panama. Spanning over five decades, this model has been instrumental in evaluating the in vivo efficacy and pharmacokinetics of a wide array of candidate antimalarial drugs, whether used singly or in combination. The animal model could be infected with drug-resistant and susceptible Plasmodium falciparum and Plasmodium vivax strains that follow a characteristic and reproducible course of infection, remarkably like human untreated and treated infections. Over the years, the model has enabled the evaluation of several synthetic and semisynthetic endoperoxides, for instance, artelinic acid, artesunate, artemether, arteether, and artemisone. These compounds have been evaluated alone and in combination with long-acting partner drugs, commonly referred to as artemisinin-based combination therapies, which are recommended as first-line treatment against uncomplicated malaria. Further, the model has also supported the evaluation of the primaquine analog tafenoquine against blood stages of P. vivax, contributing to its progression to clinical trials and eventual approval. Besides, the P. falciparum/Aotus model at GML has also played a pivotal role in exploring the biology, immunology, and pathogenesis of malaria and in the characterization of drug-resistant P. falciparum and P. vivax strains. This minireview offers a historical overview of the most significant contributions made by the Panamanian owl monkey (Aotus lemurinus lemurinus) to malaria chemotherapy research.

Harnessing cholesterol uptake of malaria parasites for therapeutic applications.

Parasites, such as the malaria parasite P. falciparum, are critically dependent on host nutrients. Interference with nutrient uptake can lead to parasite death and, therefore, serve as a successful treatment strategy. P. falciparum parasites cannot synthesise cholesterol, and instead source this lipid from the host. Here, we tested whether cholesterol uptake pathways could be 'hijacked' for optimal drug delivery to the intracellular parasite. We found that fluorescent cholesterol analogues were delivered from the extracellular environment to the intracellular parasite. We investigated the uptake and inhibitory effects of conjugate compounds, where proven antimalarial drugs (primaquine and artesunate) were attached to steroids that mimic the structure of cholesterol. These conjugated antimalarial drugs improved the inhibitory effects against multiple parasite lifecycle stages, multiple parasite species, and drug-resistant parasites, whilst also lowering the toxicity to human host cells. Steroids with introduced peroxides also displayed antimalarial activity. These results provide a proof-of-concept that cholesterol mimics can be developed as a drug delivery system against apicomplexan parasites with the potential to improve drug efficacy, increase therapeutic index, and defeat drug resistance.

Development, characterization, and evaluation of withaferin-A and artesunate-loaded pH-responsive acetal-dextran polymeric nanoparticles for the management of malaria.

Artemisinin and its derivatives have been commonly used to treat malaria. However, the emergence of resistance against artemisinin derivatives has posed a critical challenge in malaria management. In the present study, we have proposed a combinatorial approach, utilizing pH-responsive acetal-dextran nanoparticles (Ac-Dex NPs) as carriers for the delivery of withaferin-A (WS-3) and artesunate (Art) to improve treatment efficacy of malaria. The optimized WS-3 and Art Ac-Dex NPs demonstrated enhanced pH-responsive release profiles under parasitophorous mimetic conditions (pH 5.5). Computational molecular modeling reveals that Ac-Dex's polymeric backbone strongly interacts with merozoite surface protein-1 (MSP-1), preventing erythrocyte invasion. In-vitro antimalarial activity of drug-loaded Ac-Dex NPs reveals a 1-1.5-fold reduction in IC50 values compared to pure drug against the 3D7 strain of Plasmodium falciparum. Treatment with WS-3 Ac-Dex NPs (100 mg/kg) and Art Ac-Dex NPs (30 mg/kg) to Plasmodium berghei-infected mice resulted in 78.11 % and 100 % inhibition of parasitemia. Notably, the combination therapy comprised of Art and WS-3 Ac-Dex NPs achieved complete inhibition of parasitemia even at a half dose of Art, indicating the synergistic potential of the combinations. However, further investigations are necessary to confirm the safety and effectiveness of WS-3 and Art Ac-Dex NPs for their successful clinical implications.

Artesunate attenuates isoprenaline induced cardiac hypertrophy in rats via SIRT1 inhibiting NF-κB activation.

Cardiac Hypertrophy is an adaptive response of the body to physiological and pathological stimuli, which increases cardiomyocyte size, thickening of cardiac muscles and progresses to heart failure. Downregulation of SIRT1 in cardiomyocytes has been linked with the pathogenesis of cardiac hypertrophy. The present study aimed to investigate the effect of Artesunate against isoprenaline induced cardiac hypertrophy in rats via SIRT1 inhibiting NF-κB activation. Experimental cardiac hypertrophy was induced in rats by subcutaneous administration of isoprenaline (5 mg/kg) for 14 days. Artesunate was administered simultaneously for 14 days at a dose of 25 mg/kg and 50 mg/kg. Artesunate administration showed significant dose dependent attenuation in mean arterial pressure, electrocardiogram, hypertrophy index and left ventricular wall thickness compared to the disease control group. It also alleviated cardiac injury biomarkers and oxidative stress. Histological observation showed amelioration of tissue injury in the artesunate treated groups compared to the disease control group. Further, artesunate treatment increased SIRT1 expression and decreased NF-kB expression in the heart. The results of the study show the cardioprotective effect of artesunate via SIRT1 inhibiting NF-κB activation in cardiomyocytes.

Artesunate alleviates Sjögren's Syndrome by inhibiting the interferon-α signaling in plasmacytoid dendritic cells via TLR-MyD88-IRF7.

Sjögren's syndrome (SS) is an autoimmune disease in which the salivary glands (SGs) and the lacrimal glands (LGs) are affected by lymphocytic infiltration and inflammation. It has been reported that interferon-α (IFN-α) released by plasmacytoid dendritic cells (pDCs) contribute to the pathology of SS, and ART has been shown to effectively ameliorates SS. Despite the current research endeavors, the mechanism of how ART works in the treatment of SS remains to be fully elucidated. Whether ART can treat SS by inhibiting IFN-α remains unclear. This hypothesis was tested both in vivo and in vitro settings during the study. The SS model mice, which were treated with ART, showed amelioration in symptoms related to dryness. RNA-seq analysis revealed strong anti-IFN-α signaling response upon ART treatment. Additional in vitro studies provided further confirmation that the application of ART inhibits the MyD88 protein expression and the nuclear translocation of IRF7. This suggests that the intervention of ART in the TLR-MyD88-IRF7 pathway plays a role in the therapeutic approach for SS. In summary, this study highlighted the therapeutic potential of ART in SS and ART inhibited the IFN-α signaling in pDCs via the TLR-MyD88-IRF7 pathway.

Artesunate alleviates sepsis-induced liver injury by regulating macrophage polarization via the lncRNA MALAT1/PTBP1/IFIH1 axis.

BACKGROUND: The present study aimed to explore the regulatory effects of artesunate on macrophage polarization in sepsis. METHODS: Cell models and mice models were established using lipopolysaccharide (LPS), followed by treatment with various concentrations of artesunate. The phenotype of the macrophages was determined by flow cytometry. RNA immunoprecipitation was used to confirm the binding between MALAT1 and polypyrimidine tract-binding protein 1 (PTBP1), as well as between PTBP1 and interferon-induced helicase C domain-containing protein 1 (IFIH1). RESULTS: Treatment with artesunate inhibited M1 macrophage polarization in Kupffer cells subjected to LPS stimulation by downregulating MALAT1. Furthermore, MALAT1 abolished the inhibitory effect of artesunate on M1 macrophage polarization by recruiting PTBP1 to promote IFIH. In vivo experiments confirmed that artesunate alleviated septic liver injury by affecting macrophage polarization via MALAT1. CONCLUSION: The present study showed that artesunate alleviates LPS-induced sepsis in Kupffer cells by regulating macrophage polarization via the lncRNA MALAT1/PTBP1/IFIH1 axis.

Efficacy and safety of single-dose artesunate plus sulfalene/pyrimethamine combined with praziquantel for the treatment of children with Schistosoma mansoni or Schistosoma haematobium in western Kenya: a randomised, open-label controlled trial.

BACKGROUND: Reliance on praziquantel for the treatment and control of schistosomiasis is likely to facilitate the emergence of drug resistance. Combination therapy targeting adult and juvenile schistosome worms is urgently needed to improve praziquantel efficacy and delay the potential development of drug resistance. We assessed the efficacy and safety of single-dose praziquantel combined with single-dose artesunate plus sulfalene-pyrimethamine in the treatment of Kenyan children with schistosomiasis. METHODS: This was an open-label, randomised clinical trial involving 426 school-aged children (7-15 years old) diagnosed with Schistosoma mansoni (by Kato-Katz) or S. haematobium (by urine filtration). They were randomly assigned (1:1:1) to receive a single dose of praziquantel (40 mg/kg), a single dose of artesunate plus sulfalene-pyrimethamine (12 mg/kg artesunate) or combination therapy using a single dose of praziquantel (40 mg/kg) combined with a single dose of artesunate plus sulfalene-pyrimethamine (12 mg/kg artesunate). The primary outcome was cure and egg reduction rates at 6 weeks post-treatment in the available case population. Adverse events were assessed within 3 h after treatment. RESULTS: Of the 426 children enrolled, 135 received praziquantel, 150 received artesunate plus sulfalene-pyrimethamine, and 141 received combination therapy. Outcome data were available for 348 (81.7%) children. For S. mansoni-infected children (n = 335), the cure rates were 75.6%, 60.7%, and 77.8%, and the egg reduction rates were 80.1%, 85.0%, and 88.4% for praziquantel, artesunate plus sulfalene-pyrimethamine, and combination therapy, respectively. For S. haematobium-infected children (n = 145), the corresponding cure rates were 81.4%, 71.1%, and 82.2%, and the egg reduction rates were 95.6%, 97.1%, and 97.7%, respectively. Seventy-one (16.7%) children reported mild-intensity adverse events. The drugs were well tolerated and no serious adverse events were reported. CONCLUSIONS: A single oral dose of praziquantel combined with artesunate plus sulfalene-pyrimethamine cured a high proportion of children with S. haematobium but did not significantly improve the treatment efficacy for either urinary or intestinal schistosomiasis. Sequential administration of praziquantel and artesunate plus sulfalene-pyrimethamine may enhance the efficacy and safety outcomes.

Late Parasitological Failure and Subsequent Isolated Gametocytemia of Uncomplicated Plasmodium falciparum Malaria in a Returned Traveler From Ghana, 2023.

Herein, we report a case of uncomplicated falciparum malaria with late parasitological failure in a 45-year-old businessman returning from Ghana. The patient visited the emergency department with high fever, headache, and dizziness. He traveled without antimalarial chemoprophylaxis. Laboratory tests led to the diagnosis of uncomplicated falciparum malaria with an initial density of 37,669 parasites per µL of blood (p/µL). The patient was treated with intravenous artesunate followed by atovaquone/proguanil. He was discharged with improved condition and decreased parasite density of 887 p/µL. However, at follow-up, parasite density increased to 7,630 p/µL despite the absence of any symptoms. Suspecting treatment failure, the patient was administered intravenous artesunate and doxycycline for seven days and then artemether/lumefantrine for three days. Blood smear was negative for asexual parasitemia after re-treatment but positive for gametocytemia until day 101 from the initial diagnosis. Overall, this case highlights the risk of late parasitological failure in patients with imported uncomplicated falciparum malaria.

Novel artesunate and isatin hybrid CT3-1 suppresses collagen-induced arthritis through abrogating dendritic cell chemotaxis-induced by CCR5.

BACKGROUND: Chemotaxis and trafficking of dendritic cells (DCs) induced by cytokine receptors are crucial steps in rheumatoid arthritis (RA) pathogenesis. C-C chemokine receptor type 5 (CCR5) plays a key role in DC movement and has been implicated in multitudinous inflammatory and immunology diseases. Thus, targeting CCR5 to suppress DC chemotaxis is considered as a potential strategy for the management of RA. METHODS: Herein, we first synthesized a new hybrid named CT3-1 which based on artesunate and isatin. Besides, we studied the regulating effectiveness of CT3-1 on bone marrow-derived DCs (BMDCs) and on collagen-induced arthritis (CIA) through RNA-seq analysis, cell function experiments in vitro and mice model in vivo. RESULTS: The results shown that CT3-1 mainly reduced CCR5 expression of immature BMDCs and importantly inhibited immature BMDC migration induced by CCR5 in vitro, with no or minor influence on other functions of DCs, such as phagocytosis and maturation. In the mouse model, CT3-1 relieved arthritis severity and inhibited CIA development. Furthermore, CT3-1 intervention decreased the expression of CCR5 in DCs and reduced the proportion of DCs in the peripheral blood of CIA mice. CONCLUSIONS: Our findings suggest that CCR5-induced chemotaxis and trafficking of immature DCs are important in RA. Targeting CCR5 and inhibiting immature DC chemotaxis may provide a novel choice for the treatment of RA and other similar autoimmune diseases. Moreover, we synthesized a new hybrid compound CT3-1 that could inhibit immature DC trafficking and effectively relieve RA by directly reducing the CCR5 expression of immature DCs.

Exploring pectin-casein micelles as novel carriers for oral drug delivery of artesunate in the treatment of systemic lupus erythematosus.

The oral route of administration is considered the optimal choice for treating chronic diseases due to its convenience and non-invasiveness, which can help prevent physical and mental harm to patients undergoing long-term treatment. However, challenges such as safety, gastrointestinal stability, and bioavailability of oral drugs often limit their effectiveness. Natural biomacromolecule micelles, known for their safety, stability, biocompatibility, and diverse functions, have emerged as promising carriers for oral treatment of chronic diseases like systemic lupus erythematosus (SLE) with fat-soluble drugs. This study introduces an innovative approach by developing an oral delivery system using chemically synthesized natural biomacromolecules to load artesunate for treating SLE. By synthesizing amphiphilic polymer micelles from pectin and casein through a carbodiimide reaction, a more stable structure is achieved. The hydrophobic core of these micelles encapsulates artesunate, resulting in the formation of an oral delivery system (PC-AS) with several advantages, including high drug loading and encapsulation efficiency, small particle size, negative potential, strong stability in the gastrointestinal tract, low toxicity and side effects, strong adhesion in the small intestine, and high bioavailability. These advantages facilitate efficient absorption of artesunate in the gastrointestinal tract, leading to improved bioavailability and effective alleviation of SLE-like symptoms in MRL/lpr mice. By utilizing chemically synthesized natural macromolecular micelles for delivering artesunate in the treatment of SLE, this study overcomes the oral barriers associated with the original drug and presents a novel solution for the long-term oral treatment of chronic diseases.

3D printing of multi-unit gastro-retentive tablets for the pulsatile release of artesunate.

Pulsatile drug delivery is hardly achieved by conventional gastro-retentive dosage forms. Artesunate as a typical anti-malaria medicine needs oral pulsatile release. Here, artesunate-loaded pulsatile-release multi-unit gastro-retentive tablets (APGTs) were prepared with a semi-solid extrusion three-dimensional (3D) printing method. An APGT was composed of three units: artesunate-loaded immediate and delayed release units and a block unit. The matrix of the immediate/delayed release units consisted of polyvinylpyrrolidone (PVP) K30 and croscarmellose sodium, which improved the rapid release of artesunate when contacting water. The block unit consisted of octadecanol, hydroxypropyl methyl cellulose K15M, PVP K30, and poloxamer F68. APGTs showed multi-phase release in simulated gastric liquids (SGLs). The first immediate release phase continued for 1 h followed by a long block phase for 7 h. The second rapid release phase was initiated when the eroded holes in the block unit extended to the inner delayed release unit, and this phase continued for about 14 h. Low-density APGTs could ensure their long-term floating in the stomach. Oral APGTs remained in the rabbit stomach for about 20 h. 3D printing provides a new strategy for the preparation of oral pulsatile-release tablets.

Differential Anti-Fibrotic and Remodeling Responses of Human Dermal Fibroblasts to Artemisia sp., Artemisinin, and Its Derivatives.

Fibrosis is a ubiquitous pathology, and prior studies have indicated that various artemisinin (ART) derivatives (including artesunate (AS), artemether (AM), and dihydroartemisinin (DHA)) can reduce fibrosis in vitro and in vivo. The medicinal plant Artemisia annua L. is the natural source of ART and is widely used, especially in underdeveloped countries, to treat a variety of diseases including malaria. A. afra contains no ART but is also antimalarial. Using human dermal fibroblasts (CRL-2097), we compared the effects of A. annua and A. afra tea infusions, ART, AS, AM, DHA, and a liver metabolite of ART, deoxyART (dART), on fibroblast viability and expression of key fibrotic marker genes after 1 and 4 days of treatment. AS, DHA, and Artemisia teas reduced fibroblast viability 4 d post-treatment in up to 80% of their respective controls. After 4 d of treatment, AS DHA and Artemisia teas downregulated ACTA2 up to 10 fold while ART had no significant effect, and AM increased viability by 10%. MMP1 and MMP3 were upregulated by AS, 17.5 and 32.6 fold, respectively, and by DHA, 8 and 51.8 fold, respectively. ART had no effect, but A. annua and A. afra teas increased MMP3 5 and 16-fold, respectively. Although A. afra tea increased COL3A1 5 fold, MMP1 decreased >7 fold with no change in either transcript by A. annua tea. Although A. annua contains ART, it had a significantly greater anti-fibrotic effect than ART alone but was less effective than A. afra. Immunofluorescent staining for smooth-muscle α-actin (α-SMA) correlated well with the transcriptional responses of drug-treated fibroblasts. Together, proliferation, qPCR, and immunofluorescence results show that treatment with ART, AS, DHA, and the two Artemisia teas yield differing responses, including those related to fibrosis, in human dermal fibroblasts, with evidence also of remodeling of fibrotic ECM.

Exploring the neuroprotective role of artesunate in mouse models of anti-NMDAR encephalitis: insights from molecular mechanisms and transmission electron microscopy.

BACKGROUND: The pathway involving PTEN-induced putative kinase 1 (PINK1) and PARKIN plays a crucial role in mitophagy, a process activated by artesunate (ART). We propose that patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis exhibit insufficient mitophagy, and ART enhances mitophagy via the PINK1/PARKIN pathway, thereby providing neuroprotection. METHODS: Adult female mice aged 8-10 weeks were selected to create a passive transfer model of anti-NMDAR encephalitis. We conducted behavioral tests on these mice within a set timeframe. Techniques such as immunohistochemistry, immunofluorescence, and western blotting were employed to assess markers including PINK1, PARKIN, LC3B, p62, caspase3, and cleaved caspase3. The TUNEL assay was utilized to detect neuronal apoptosis, while transmission electron microscopy (TEM) was used to examine mitochondrial autophagosomes. Primary hippocampal neurons were cultured, treated, and then analyzed through immunofluorescence for mtDNA, mtROS, TMRM. RESULTS: In comparison to the control group, mitophagy levels in the experimental group were not significantly altered, yet there was a notable increase in apoptotic neurons. Furthermore, markers indicative of mitochondrial leakage and damage were found to be elevated in the experimental group compared to the control group, but these markers showed improvement following ART treatment. ART was effective in activating the PINK1/PARKIN pathway, enhancing mitophagy, and diminishing neuronal apoptosis. Behavioral assessments revealed that ART ameliorated symptoms in mice with anti-NMDAR encephalitis in the passive transfer model (PTM). The knockdown of PINK1 led to a reduction in mitophagy levels, and subsequent ART intervention did not alleviate symptoms in the anti-NMDAR encephalitis PTM mice, indicating that ART's therapeutic efficacy is mediated through the activation of the PINK1/PARKIN pathway. CONCLUSIONS: At the onset of anti-NMDAR encephalitis, mitochondrial damage is observed; however, this damage is mitigated by the activation of mitophagy via the PINK1/PARKIN pathway. This regulatory feedback mechanism facilitates the removal of damaged mitochondria, prevents neuronal apoptosis, and consequently safeguards neural tissue. ART activates the PINK1/PARKIN pathway to enhance mitophagy, thereby exerting neuroprotective effects and may achieve therapeutic goals in treating anti-NMDAR encephalitis.

Antitumour effects of artesunate via cell cycle checkpoint controls in human oesophageal squamous carcinoma cells.

Artesunate (ART), an effective antimalarial semisynthetic derivative of artemisinin, exhibits antitumour properties, but the mechanism(s) involved remain elusive. In this study, we investigated the antitumour effects of ART on human oesophageal squamous cell carcinoma (ESCC) cell lines. Treatment of ESCC cell lines with ART resulted in the production of excessive reactive oxygen species (ROS) that induced DNA damage, reduced cell proliferation and inhibited clonogenicity via G1-S cell cycle arrest and/or apoptosis in vitro. The administration of ART to nude mice with ESCC cell xenografts inhibited tumour formation in vivo. However, the cytotoxicity of ART strongly differed among the ESCC cell lines tested. Transcriptomic profiling revealed that although the expression of large numbers of genes in ESCC cell lines was affected by ART treatment, these genes could be functionally clustered into pathways involved in regulating cell cycle progression, DNA metabolism and apoptosis. We revealed that p53 and Cdk4/6-p16-Rb cell cycle checkpoint controls were critical determinants required for mediating ART cytotoxicity in ESCC cell lines. Specifically, KYSE30 cells with p53Mut/p16Mut were the most sensitive to ART, KYSE150 and KYSE180 cells with p53Mut/p16Nor exhibited intermediate responses to ART, and Eca109 cells with p53Nor/p16Nor exhibited the most resistance to ATR. Consistently, perturbation of p53 expression using RNA interference (RNAi) and/or Cdk4/6 activity using the inhibitor palbociclib altered ART cytotoxicity in KYSE30 cells. Given that the p53 and Cdk4/6-cyclin D1-p16-Rb genes are commonly mutated in ESCC, our results potentially shed new light on neoadjuvant chemotherapy strategies for ESCC.

Lacrimal gland Alterations and the Effect of artesunate on experimental induced diabetes rat models and related mechanisms.

Diabetic patients are at high risk of developing lacrimal gland dysfunction, and the antimalarial drug artesunate (ART) was recently used to induce experimental-induced diabetes mellitus. This study's objective is to investigate the lacrimal gland alteration and the effect of ART on experimentally induced diabetes rat models and its related mechanisms. Forty rats were divided into five groups (8 rats/group): healthy control group (HC), diabetic group (DM), 50 mg/kg ART intervention diabetic group [DM + ART (50 mg/kg)], 100 mg/kg ART intervention diabetic group [DM + ART (100 mg/kg)] and 6 U/kg Insulin intervention diabetic group (DM + INS). The morphology of the eyeball and lacrimal gland tissues was determined using hematoxylin and eosin staining. In addition, external lacrimal glands were harvested for electronic microscopic examination, NFκB1, and TNF-α protein expression evaluation by immunohistochemistry and mRNA expression analysis by RT-PCR. Histopathological and ultrastructural changes suggest ART intervention has an improved structural effect. Protein expression of NFκB1 in the DM + ART (100 mg/kg) group was decreased. TNF-α significantly decreased in the DM + ART (50 mg/kg) and insulin groups. We concluded that ART improves structural changes in a lacrimal gland in diabetic rats. The present study provides further evidence of the therapeutic effect of ART on the lacrimal gland of diabetic rats by decreasing the expression of NFκB1 and TNF-α.