Immunosuppressive effect of artemisinin and hydroxychloroquine combination therapy on IgA nephropathy via regulating the differentiation of CD4+ T cell subsets in rats.

Immunoglobulin A nephropathy (IgAN) is an autoimmune kidney disease with complex pathogenesis leading to end-stage renal damage. The crucial pathological characteristic in IgAN is IgA immune complexes deposition accompany with mesangial cell proliferation and mesangial matrix expansion. Artemisinin (ART) is isolated from traditional Chinese medicine Artemisia annua L. Hydroxychloroquine (HCQ) is a classical antimalarial drug used to treat autoimmune diseases. Both of them possess immunosuppressive, immunomodulatory and anti-inflammatory features. The aim of this study was to investigate the pharmacological effects of ART combined with HCQ (AH) and explore the underlying mechanisms in IgAN. In vivo, our results showed that AH could significantly improve kidney dysfunction, decrease mesangial matrix expansion as well as immune complexes in mesangial area visualized by H&E and PAS staining. The depositions of IgA immune complexes and complement 3 (C3) were obviously reduced after AH treatment by immunofluorescence. Interestingly, the morphology of kidney and spleen was significantly swelled but reverted by AH in IgAN rats. Further mechanistic study showed that the higher proportions of the Th2 and Th17 cells were reduced but the lower differentiation of Th1 and Treg cells subsets were promoted by AH. Taken together, this study demonstrated that there was an immunosuppressive effect of AH therapy on IgAN rats via regulating the differentiation of CD4+ T cell subsets, which provided an alternative approach for IgAN treatment.

Artesunate ameliorates severe acute pancreatitis (SAP) in rats by inhibiting expression of pro-inflammatory cytokines and Toll-like receptor 4.

Severe acute pancreatitis (SAP) is a severe clinical condition with significant morbidity and mortality. Multiple organs dysfunction (MOD) is the leading cause of SAP-related death. The over-release of pro-inflammatory cytokines such as IL-1ß, IL-6, and TNF-α is the underlying mechanism of MOD; however, there is no effective agent against the inflammation. Herein, artesunate (AS) was found to increase the survival of SAP rats significantly when injected with 3.5% sodium taurocholate into the biliopancreatic duct in a retrograde direction, improving their pancreatic pathology and decreasing serum amylase and pancreatic lipase activities along with substantially reduced pancreatic IL-1ß and IL-6 release. In vitro, AS-pretreatment strongly inhibited IL-1ß and IL-6 release and their mRNA expressions in the pancreatic acinar cells treated with lipopolysaccharide (LPS) but exerted little effect on TNF-α release. Additionally, AS reduced the mRNA expressions of Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) p65 as well as their protein expressions in the pancreatic acinar cells. In conclusion, our results demonstrated that AS could significantly protect SAP rats, and this protection was related to the reduction of digestive enzyme activities and pro-inflammatory cytokine expressions via inhibition of TLR4/NF-κB signaling pathway. Therefore, AS may be considered as a potential therapeutic agent against SAP.

Dihydroartemisinin alleviates bile duct ligation-induced liver fibrosis and hepatic stellate cell activation by interfering with the PDGF-ßR/ERK signaling pathway.

Liver fibrosis represents a frequent event following chronic insult to trigger wound healing responses in the liver. Activation of hepatic stellate cells (HSCs), which is a pivotal event during liver fibrogenesis, is accompanied by enhanced expressions of a series of marker proteins and pro-fibrogenic signaling molecules. Artemisinin, a powerful antimalarial medicine, is extracted from the Chinese herb Artemisia annua L., and can inhibit the proliferation of cancer cells. Dihydroartemisinin (DHA), the major active metabolite of artemisinin, is able to attenuate lung injury and fibrosis. However, the effect of DHA on liver fibrosis remains unclear. The aim of this study was to investigate the effect of DHA on bile duct ligation-induced injury and fibrosis in rats. DHA improved the liver histological architecture and attenuated collagen deposition in the fibrotic rat liver. Experiments in vitro showed that DHA inhibited the proliferation of HSCs and arrested the cell cycle at the S checkpoint by altering several cell-cycle regulatory proteins. Moreover, DHA reduced the protein expressions of a-SMA, α1 (I) collagen and fibronectin, being associated with interference of the platelet-derived growth factor ß receptor (PDGF-ßR)-mediated ERK pathway. These data collectively revealed that DHA relieved liver fibrosis possibly by targeting HSCs via the PDGF-ßR/ERK pathway. DHA may be a therapeutic antifibrotic agent for the treatment of hepatic fibrosis.

Efficacy of Artemisia annua polysaccharides as an adjuvant to hepatitis C vaccination.

The traditional Chinese medicine Artemisia annua can prevent and treat hepatitis following an unclear mechanism. The aim of this study was to evaluate the effects of A. annua polysaccharides (AAP) on hepatitis C virus (HCV). A pcDNA3.1/NS3 expression vector was constructed. Ninety female BALB/c mice were randomly divided into six groups: high-dose AAP (1 mg/mL) + HCV/NS3 plasmid; middle-dose AAP (0.5 mg/mL) + HCV/NS3 plasmid; low-dose AAP (0.1 mg/mL) + HCV/NS3 plasmid; HCV/NS3 plasmid; high-dose AAP (1 mg/mL); normal saline control (N = 15). Except the control group and the high-dose AAP group, other groups were inoculated with 50 µg pcDNA3.1-HCV/NS3 plasmid. Serum antigenic-specific antibody was detected after the last immunization, and the levels of secreted IFN-γ and IL-4 were measured. pcDNA3.1/NS3 plasmid was successfully constructed, and the extracted product contained HCV/NS3 sequence. Compared with single inoculation with HCV/NS3 DNA vaccine, the specific antibody levels induced by middle-dose AAP plus HCV/NS3 DNA vaccine were significantly different in weeks 1, 3 and 5 (P < 0.05). However, there were no significant differences in the antibody levels induced by high-dose and low-dose AAP as adjuvant compared with those of single inoculation with DNA vaccine (P > 0.05). The level of serum IFN-γ secretion was significantly higher than that of IL-4 secretion. Compared with the single HCV/NS3 DNA vaccine group, AAP plus HCV/NS3 DNA vaccine groups had significant increased IFN-γ levels (P < 0.05), but the IL-4 levels were not significantly different among these groups (P > 0.05). AAP, as the adjuvant of HCV/NS3 DNA vaccine, can widely regulate the humoral immunity and cellular immune function of normal and cyclophosphamide-induced immunocompromised mice. AAP can promote IFN-γ secretion probably by inducing Th1-type cellular immune response.

Dihydroartemisinin inhibits activation of the Toll-like receptor 4 signaling pathway and production of type I interferon in spleen cells from lupus-prone MRL/lpr mice.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by various immunological abnormalities. Dihydroartemisinin (DHA), a metabolite of artemisinin, has been recently reported to exhibit immunosuppressive properties. The present study aims to determine the effects of DHA on spleen cell activation triggered by lipopolysaccharide (LPS) and investigate the effects of DHA on LPS-induced activation of the Toll-like receptor 4 (TLR4)/interferon regulatory factor (IRF) signaling pathway. Spleen cells from lupus-prone MRL/lpr mice were isolated, prepared and cultured. Cells were treated with LPS alone or LPS with DHA, and spleen cell proliferation was analyzed using MTS assay. Protein expressions of TLR4, IRF3, and IRF7 were analyzed by Western blot. IRF3 phosphorylation was also determined. Gene expression levels of IFN-α and IFN-ß were measured using real-time PCR, and protein levels in cells' supernatants were determined by ELISA. DHA was found to inhibit LPS-induced spleen cell proliferation, decrease LPS-induced protein expression of TLR4, and inhibit IRF3 phosphorylation. Furthermore, LPS significantly induced IRF3 expression and slightly increased IRF7 expression in the nucleus of spleen cells, which was accompanied by enhanced IFN-α and IFN-ß production. DHA inhibited the effects of LPS in spleen cells of MRL/lpr mice. Taken together, the data obtained reveal that DHA inhibits LPS-induced cell activation possibly by suppressing the TLR4/IRF/IFN pathway in spleen cells of MRL/lpr mice. These data suggest that DHA has the potential therapeutic utility for the treatment of SLE.

AaORA, a trichome-specific AP2/ERF transcription factor of Artemisia annua, is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea.

· Six transcription factors of APETALA2/ethylene-response factor (AP2/ERF) family were cloned and analyzed in Artemisia annua. Real-time quantitative polymerase chain reaction (RT-Q-PCR) showed that AaORA exhibited similar expression patterns to those of amorpha-4,11-diene synthase gene (ADS), cytochrome P450-dependent hydroxylase gene (CYP71AV1) and double bond reductase 2 gene (DBR2) in different tissues of A. annua. · AaORA is a trichome-specific transcription factor, which is expressed in both glandular secretory trichomes (GSTs) and nonglandular T-shaped trichomes (TSTs) of A. annua. The result of subcellular localization shows that AaORA is targeted to the nuclei and the cytoplasm. · Overexpression and RNA interference (RNAi) of AaORA in A. annua regulated, positively and significantly, the expression levels of ADS, CYP71AV1, DBR2 and AaERF1. The up-regulated or down-regulated expression levels of these genes resulted in a significant increase or decrease in artemisinin and dihydroartemisinic acid. The results demonstrate that AaORA is a positive regulator in the biosynthesis of artemisinin. · Overexpression of AaORA in Arabidopsis thaliana increased greatly the transcript levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2), HEVEIN-LIKE PROTEIN (HEL) and BASIC CHITINASE (B-CHI). After inoculation with Botrytis cinerea, the phenotypes of AaORA overexpression in A. thaliana and AaORA RNAi in A. annua demonstrate that AaORA is a positive regulator of disease resistance to B. cinerea.

Immunosuppressive effect of ethanol extract of Artemisia annua on specific antibody and cellular responses of mice against ovalbumin.

Artemisia annua has been widely used to treat autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis in traditional Chinese medicine. In this study, the ethanol extract of A. annua (EEAA) was evaluated for the immunosuppressive potentials on mice splenocyte proliferation in vitro, and the specific antibody and cellular immune responses in the ovalbumin (OVA)-immunized mice. EEAA significantly suppressed concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated splenocyte proliferation in vitro in a concentration-dependent manner. EEAA also significantly suppressed Con A-, LPS- and OVA-induced splenocyte proliferation in the OVA-immunized mice in a dose-dependent manner. Meanwhile, the OVA-specific serum IgG, IgG1 and IgG2b antibody levels in the OVA-immunized mice were markedly reduced by EEAA. The results suggest that EEAA could suppress the cellular and humoral response in mice. This study provided evidence to understand the therapeutic effects of A. annua for treatment of some autoimmune diseases and an immunosuppressive natural products to further researches to be developed as immunosuppressant.