Daphnetin induces apoptosis in fibroblast-like synoviocytes from collagen-induced arthritic rats mainly via the mitochondrial pathway.

Rheumatoid arthritis (RA) is a chronic, symmetric, systemic autoimmune disease. Because insufficient apoptosis of fibroblast-like synoviocytes (FLS) is an important characteristic of RA, promoting apoptosis is considered a potential therapeutic tool for treating RA. We have previously found that daphnetin (7,8-dihydroxycoumarin, DAP) has a pro-apoptotic effect on fibroblast-like synoviocytes from collagen-induced arthritis (CIA) rats. In the present study, we further investigated the mechanisms of DAP-induced apoptosis in CIA-FLS. CIA-FLS were incubated with DAP for 48 h in the presence or absence of caspase inhibitors, including inhibitors of caspase-3, caspase-8, or caspase-9 or a pan-caspase inhibitor; then, a series of experiments were performed to evaluate the mechanisms of DAP-induced apoptosis. Our results showed that DAP markedly decreased cell viability and induced the apoptosis of CIA-FLS along with typical morphological and ultrastructural changes; moreover, DAP increased FasL, cytochrome c (Cyt-c), Bax, caspase-3, caspase-8, and caspase-9 mRNA expression and Bax, caspase-3, caspase-8, and caspase-9 protein expression. In contrast, DAP decreased Bcl-2 mRNA and protein expression and promoted the release of Cyt-c from the mitochondria into the cytosol; these effects were attenuated to varying degrees by pre-treatment with caspase inhibitors, especially with caspase-3 or caspase-9 inhibitors or a pan-caspase inhibitor. In conclusion, the current findings demonstrate that the DAP-induced apoptosis of CIA-FLS occurred mainly via a caspase-dependent pathway, in particular the mitochondrial pathway, and that the Bax/Bcl-2 ratio was involved in this process. Thus, DAP may be a potential therapeutic agent for RA.

Effects of daphnetin on the autophagy signaling pathway of fibroblast-like synoviocytes in rats with collagen-induced arthritis (CIA) induced by TNF-α.

Daphnetin (DAP), an active ingredient extracted from Daphne odora, has pharmacological effects such as anti-inflammatory, antioxidation and anti-tumor properties. The current study aims to investigate the relationship between the anti-rheumatoid effect of DAP and the inhibition of both the PI3K/AKT/mTOR and autophagy signaling pathways. DAP inhibited the proliferation of CIA-FLS in a dose-dependent manner and induce apoptosis, accelerated the G1/G0 phase and inhibited the S phase. DAP reduced the phosphorylation of AKT and mTOR and the expression of Atg5, Beclin-1 and LC3-II/LC3-I in CIA-FLS induced by TNF-α. DAP also reduced the inflammatory response in CIA-FLS induced by TNF-α by inhibiting the cytokine expression of TNF-α, IL-6, TGF-ß, IL-17, and INF-γ and promoting IL-10 expression. Overall, DAP inhibited the proliferation of CIA-FLS by down-regulating the PI3K/AKT/mTOR signaling pathway and inhibited autophagy in order to induces apoptosis, which may be potential therapeutic approach in treatment of RA.

CD38 Deficiency Downregulates the Onset and Pathogenesis of Collagen-Induced Arthritis through the NF-κB Pathway.

AIM: The RelB gene plays an important role in guiding the progression of arthritis. We have previously demonstrated that the expression of the RelB gene is decreased significantly in bone marrow DCs of CD38-/- mice. In this study, we demonstrate that the cluster of the differentiation (CD38) gene could be a potentially therapeutic target for autoimmune arthritis. METHOD: Collagen-induced arthritis (CIA) models were generated with both the wild-type (WT) C57BL/6 and CD38-/- mice. The expression of the RelB gene and maturation of bone marrow-derived dendritic cells (DCs) from the WT and CD38-/- mice were detected. Antigen-specific T cell responses, joint damage, and expression of proinflammatory cytokines were assessed. The effects of the Nuclear Factor Kappa B (NF-κB) transcription factor and its mechanisms were characterized. RESULTS: We demonstrated that in CD38-/- mice, the expression of the RelB gene and major histocompatibility complex II (MHC II) was decreased, accompanied with the inhibited T cell reaction in a mixed lymphocyte reaction (MLR) in bone marrow-derived DCs. Compared to the serious degeneration of the cartilage and the enlarged gap of the cavum articular in WT CIA mice, joint pathological changes of the CD38-/- CIA mice revealed marked attenuation, while the joint structures were well preserved. The preserved effects were observed by the inhibition of proinflammatory cytokines and promotion of anti-inflammatory cytokines. Furthermore, decreased phosphorylation of NF-κB was also observed in CD38-/- CIA mice. CONCLUSION: We demonstrate that CD38 could regulate CIA through NF-κB and this regulatory molecule could be a novel target for the treatment of autoimmune inflammatory joint disease.

Erratum to "Effects of daphnetin on the autophagy signaling pathway of fibroblast-like synoviocytes in rats with collagen-induced arthritis (CIA) induced by TNF-α" [Cytokine: X 1 (2019) 100015].

[This corrects the article DOI: 10.1016/j.cytox.2019.100015.].

A new cancer immunotherapy via simultaneous DC-mobilization and DC-targeted IDO gene silencing using an immune-stimulatory nanosystem.

The activity of negative immune regulatory molecules, such as indoleamine 2,3-oxygenase (IDO), significantly attenuates DC (Dendritic cells)-mediated immunotherapy. We have previously reported that knockdown of IDO using siRNA can reinstall anti-tumor immunity. However, a DC-targeted siRNA delivery system for in vivo mobilized DCs remains to be developed, while gene silencing in mobilized DCs for cancer immunotherapy has never been explored. In our study, we developed a novel DC-targeted siRNA delivery system, man-GNR-siIDO, using as a nanocarrier of siRNA specific for IDO (siIDO) and mannose (man) as a guide molecule for targeting DCs. We explored the immunostimulatory man-GNR-siIDO nano-construct in DCs mobilized by Flt3-L, a receptor-type tyrosine kinase ligand, for lung cancer immunotherapy. In vivo DC-targeted gene silencing of IDO resulted in robust anti-tumor immunity as evidenced by promoting DC maturation, up-regulating tumor antigen-specific T-cell proliferation and enhancing tumor-specific cytotoxicity. A combinatorial treatment for Lewis Lung Carcinoma (LLC)-bearing mice, with man-GNR-siIDO and Flt3-L, significantly attenuated tumor growth and delayed tumor formation, suggesting the treatment feasibility of the man-GNR-siIDO system in Flt3-L mobilized DCs in the immunotherapy of lung cancer. Therefore, our study highlights a clinical potential for a first-in-class anti-cancer immunotherapy through simultaneous DC-mobilization and DC-targeted gene silencing of IDO with man-GNR-siIDO and Flt3-L treatments.

Effects of daphnetin combined with Bcl2­siRNA on antiapoptotic genes in synovial fibroblasts of rats with collagen­induced arthritis.

The aim of the present study was to investigate the effects of daphnetin combined with B cell lymphoma 2 (Bcl2)­targeted small interfering (si)RNA (si­Bcl2) on antiapoptotic genes in fibroblast­like synoviocytes (FLS) in rats with collagen II­induced arthritis (CIA). The roles of si­Bcl2 and daphnetin were determined by measuring the expression levels of Bcl2. Protein and mRNA expression levels of Bcl2 in FLS were determined by flow cytometry and reverse transcription­quantitative polymerase chain reaction. Apoptosis of FLS was also determined by flow cytometry. It was revealed that treatment with si­Bcl2 or daphnetin alone resulted in downregulation of Bcl2 mRNA and protein expression. In addition, the mRNA expression levels of the signal transducer and activator of transcription 3 (STAT3), which transcriptionally regulates the activity of mitochondria, were reduced. The combination of si­Bcl2 and daphnetin exhibited an enhanced effect on rheumatoid arthritis FLS (RAFLS), in which the apoptotic rate was significantly higher than either treatment alone. The results suggested that si­Bcl combined with daphnetin may have an enhanced effect in promoting apoptosis of RAFLS derived from CIA rats, and a possible underlying molecular mechanism may function through the downregulation of Bcl2 expression and STAT3, which is located upstream of Bcl2 in the mitochondrial apoptotic pathway. The results of the present study are expected to provide theoretical and experimental basis for the treatment of RA and the medicinal development of daphnetin combined siRNA.

A novel GNRs-PEI/GNRs-PEI-folate for efficiently delivering siRNA.

RNA interference (RNAi) employs double-stranded RNA or siRNA (small interfering RNA) to silence gene expression in cells. The widespread use of RNAi therapeutics requires the development of clinically suitable, safe and effective delivery vehicles. PEI (Poly(ethylene imine)) carrying the positive charges has attracted considerable attention for siRNA delivery. Gold nanorods (GNRs) exhibit specially localized surface plasmon resonance when excited by the visible and near-infrared laser, which is useful for photothermal therapy. However, the toxicity derived from a large amount of the surfactant cetyltrimethylammonium bromide (CTAB) during GNR synthesis severely limits their medical applications. Here, we report the synthesis of GNRs-PEI/GNRs-PEI-folate to improve biocompatibility, siRNA delivery and photothermal therapy of GNRs. Firstly, GNRs were synthesized according to the seed-mediated template-assisted protocol. The characterization results of GNRs showed: the size was length about 218 nm and width about 26.8 nm; the Zeta potential was +38.1 mV derived from CTAB on their surface; the dipole resonance extinction spectrum peak was 752 nm which is effective for photothermal therapy in vivo. Secondly, we synthesized PEI-MUA (Mercaptoundecanoic acid) and PEI-MUA-folate based on the chemical reaction between amino group of PEI and carboxyl group of MUA or Folate. PEI-MUA or PEI-MUA-folate to replace CTAB on GNRs obtained the GNRs-MUA-PEI system or the GNRs-MUA-PEI-folate system due to the solid conjugation between the thiol group of MUA and GNRs. The products were measured using the FTIR Spectrometer, and the spectra suggest MUA-PEI or PEI-MUA-folate has successfully replaced CTAB on the surface of GNRs. Finally, GNRs-MUA-PEI was incubated with siRNA-Cy3. The unbound siRNA-Cy3 was measured the intensity of fluorescence for calculating the uploaded amount of siRNA by GNRs-MUA-PEI, and the results indicate that the uploaded percentage of siRNA is about 70%. We conclude that the GNRs-MUA-PEI system is an effective siRNA loading vehicle.

Therapeutic effect of daphnetin on the autoimmune arthritis through demethylation of proapoptotic genes in synovial cells.

BACKGROUND: We have previously reported that dephnetin is therapeutically effective in the treatment of rheumatoid arthritis (RA) in collagen-induced arthritis (CIA) rat model. However, the molecular mechanism and the effect of daphnetin on demethylating proapoptotic genes in the synovial cells remains further clarified. This study may provide a deeper insight into the medicinal application of daphnetin as a treatment for RA. METHODS: (1) The proliferation inhibition of CIA rat synovial cells was determined by an MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenyterazoliumromide) assay; (2) Methylation specific PCR (MSP) was used to measure the methylation of the proapoptotic genes DR3 (death receptor 3), PDCD5 (programmed cell death 5), FasL and p53; (3) Real time-PCR was performed to determine the mRNA expression of DR3, PDCD5, FasL, p53 and DNA methyltransferases (DNMTs) DNMT1, DNMT3a and DNMT3b; (4) Flow cytometry was applied to detect the protein expression of the DR3, PDCD5, FasL and p53; (5) The apoptotic rate of synovial cells was assessed by flow cytometry with Annexin V and propidium iodide (PI); (6) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the changes of CIA rat synovial cell structure. RESULTS: (1) In the range of 1.25 µg/mL to 40 µg/mL, daphnetin and 5-aza-dc had a dose-dependent and time-dependent degree of inhibition to the CIA rat synovial cells. (2) Daphnetin and 5-aza-dc had a demethylating role on the proapoptotic genes DR3, PDCD5, FasL and p53 of CIA rat synovial cells. (3) Daphnetin and 5-aza-dc decreased the gene expression of methyltransferases DNMT1, DNMT3a and DNMT3b, and increased expression of proapoptotic genes DR3, PDCD5, FasL and p53, which translated into an increased protein expression of DR3, PDCD5, FasL and p53. (4) Daphnetin and 5-aza-dc changed the structure of CIA rat synovial cells to show apoptotic changes and increased the rate of apoptosis. CONCLUSIONS: Daphnetin can reduce the expression of DNMT1, DNMT3a and DNMT3b, which could result in the proapoptotic genes DR3, PDCD5, FasL and p53 being demethylated. Therefore, daphnetin can increase proapoptotic gene and protein expression resulting in structural apoptotic changes and an increase in early and late CIA rat synovial cell apoptosis.

Baicalin prevents Candida albicans infections via increasing its apoptosis rate.

BACKGROUND: These experiments were employed to explore the mechanisms underlying baicalin action on Candida albicans. METHODOLOGY AND PRINCIPAL FINDINGS: We detected the baicalin inhibition effects on three isotope-labeled precursors of (3)H-UdR, (3)H-TdR and (3)H-leucine incorporation into C. albicans using the isotope incorporation technology. The activities of Succinate Dehydrogenase (SDH), cytochrome oxidase (CCO) and Ca(2)(+)-Mg(2+) ATPase, cytosolic Ca(2+) concentration, the cell cycle and apoptosis, as well as the ultrastructure of C.albicans were also tested. We found that baicalin inhibited (3)H-UdR, (3)H-TdR and (3)H-leucine incorporation into C.albicans (P<0.005). The activities of the SDH and Ca(2)(+)-Mg(2+) ATPase of C.albicans in baicalin groups were lower than those in control group (P<0.05). Ca(2+) concentrations of C. albicans in baicalin groups were much higher than those in control group (P<0.05). The ratio of C.albicans at the G0/G1 stage increased in baicalin groups in dose dependent manner (P<0.01). There were a significant differences in the apoptosis rate of C.albicans between baicalin and control groups (P<0.01). After 12-48 h incubation with baicalin (1mg/ml), C. albicans shown to be markedly damaged under transmission electron micrographs. INNOVATION AND SIGNIFICANCE: Baicalin can increase the apoptosis rate of C. albicans. These effects of Baicalin may involved in its inhibiting the activities of the SDH and Ca(2)(+)-Mg(2+) ATPase, increasing cytosolic Ca(2+) content and damaging the ultrastructure of C. albicans.

The therapeutic effects of daphnetin in collagen-induced arthritis involve its regulation of Th17 cells.

OBJECTIVES: Daphne odora var. marginata (D. marginata), an aiophyllus arbuscular plant, is one of the traditional Chinese medicines used to treat rheumatoid arthritis. This study investigated the therapeutic effects and mechanisms of daphnetin, an active monomer ingredient derived from D. marginata, on collagen-induced arthritis (CIA) in rats. METHODS: The effects of daphnetin on joint diseases were assessed by hematoxylin and eosin staining and radiographic and transmission electron microscopy. The protein and mRNA expression levels of T helper (Th)1/Th2/Th17-type cytokines in the spleen were determined by flow cytometry and quantitative real-time PCR. RESULTS: Our results showed that daphnetin significantly reduced paw swelling and was nontoxic in vivo at the tested doses. Synovial hyperplasia, joint destruction and chondrocyte degeneration in CIA rats were suppressed by daphnetin. Daphnetin treatment also reduced the levels of Th1/Th2/Th17 type cytokines in spleen lymphocytes in CIA rats. Moreover, the expression of Foxp3, which can down-regulate the activity of Th17 cells, was significantly increased in the daphnetin-treated groups. CONCLUSIONS: These results suggest that daphnetin may have therapeutic effects in down-regulating Th17-type responses in CIA rats. The beneficial effects of daphnetin on CIA may be related to its inhibition of Th17 cell priming and activation.

Role of TLR4 and TCR or BCR against baicalin-induced responses in T and B cells.

Baicalin (BA), a flavonoid compound isolated from Scutellaria baicalensis, has been shown to possess a number of pharmacological effects including antiviral, anti-inflammatory, antioxidant and immune regulation. Here, we examined its effects on human T and B cells proliferation by MTT assay and found that BA stimulated T and B cells proliferation, independently and cooperatively with Con-A (T cells) or LPS (B cells). Then, we analyzed the effects of BA treatment on the mRNA expression of Toll-like receptors (TLRs), IL-2, IFN-γ and IL-12 in T and B cells by real-time RT-PCR and attempted to observe whether blocking TLR4 had influence on mRNA expression. We found that BA treatment significantly up-regulated TLR3, 7, 8 and 9 mRNA expressions in T and B cells, IL-2 and IFN-γ in T cells and IL-12 in B cells. The increased mRNA expressions were suppressed after blocking TLR4. We further analyzed the effects of BA treatment on TCR vß and CD79 mRNA expression levels in T and B cells and explored whether blocking TCR (αß) or BCR mIgM F(ab')(2) had an influence on mRNA expression. We found that BA treatment significantly improved TCR vß and CD79 mRNA expression in T and B cells, respectively, and the improvements were all inhibited after blocking TCR (αß) or BCR mIgM F(ab')(2). Our results suggested that BA participates in innate and adaptive immune regulation by up-regulating the mRNA expression of TLRs (3, 7, 8 and 9), IL-2, IFN-γ and IL-12 in T and B cells, which is mediated by TLR4, and by improving the mRNA expression of TCR vß and CD79, which is mediated by TCR (αß) and BCR mIgM, respectively. Therefore, TLR4, TCR (αß) and BCR mIgM are all the immune receptors for BA on T and B cells.

Regulatory effect of daphnetin, a coumarin extracted from Daphne odora, on the balance of Treg and Th17 in collagen-induced arthritis.

Daphnetin extracted from Daphne odora Var. marginata contains coumarin compounds, which possess properties of analgesic and anti-inflammatory effects. In this study, we investigated the therapeutic effect of daphnetin on anti-arthritis and its role on the balance of Tregs and Th17, using a collagen-induced arthritis rat model. Collagen-induced arthritis rats were treated with daphnetin for 21 days. The therapeutic effects of daphnetin were evaluated by clinical symptoms and histopathology. The levels of Th17-, Treg-, Th2-, Th1-type cytokines in serum were determined by ELISA. The expression levels of related receptors RORγt, NF-κB, Foxp3 and CD77 in joint tissues were detected by immunohistochemistry. Our results showed that administration of daphnetin significantly alleviated the severity of the arthritis, as evidenced by the reduction of arthritis scores, suppression of the infiltration of inflammatory cells and prevention of synovial hyperplasia, thereby resulting in the joint destruction in the arthritis rats. Additionally, daphnetin treatment reduced the serum level of Th17-, Th2- and Th1-type in collagen-induced arthritis rats. Correspondingly, the expression of RORγt, NF-κB and CD77 in joint tissue of collagen-induced arthritis rats was remarkably decreased, while the expression of Foxp3 and IL-10 was remarkably increased after being administered with daphnetin. Collectively, this study demonstrated that administration of daphnetin attenuated the clinical symptoms and pathological destruction of arthritis joints. The therapeutic effects were associated with the up-regulation of Tregs, down-regulation of Th17-, Th2- and Th1-type cell responses. The results provide novel evidence that daphnetin has therapeutic effects on autoimmune arthritis through modulating the balance of Tregs and Th17.

[The genetic polymorphisms of 6 short tandem repeat loci in native Han population in Jiangxi province].

OBJECTIVE: To investigate the genetic polymorphisms of 6 short tandem repeats (STR) loci, namely, D6S1043, D2S1772, D7S3048, D22-GATA198B05, D8S1132 and D11S2368 in native Han population of Jiangxi province, China. METHODS: Two hundred and twelve blood samples of unrelated subjects of Han population in Jiangxi province were collected. Genotyping was performed by using multiplex polymerase chain reaction-polyacrylamide gel electrophoresis. RESULTS: Thirteen alleles and 52 genotypes in the D6S1043 locus, 13 alleles and 66 genotypes in D2S1772, 12 alleles and 48 genotypes in D7S3048, 11 alleles and 44 genotypes in D22-GATA198B05, 10 alleles and 38 genotypes in D8S1132, 10 alleles and 41 genotypes in D11S2368 locus, were detected in the 212 subjects. The observed heterozygosity (h) in D6S1043, D2S1772, D7S3048, D22-GATA198B05, D8S1132 and D11S2368 loci ranged from 0.8019 to 0.8774. The expected heterozygosity (H) varied from 0.8553 to 0.8896. The discriminating power (DP) ranged from 0.9559 to 0.9735. The probability of exclusion (PE) ranged from 0.7053 to 0.7751. The polymorphism information content (PIC) varied from 0.8452 to 0.8774. CONCLUSION: All the 6 loci were of Hardy-Weinberg equilibrium. The genetic polymorphism of the 6 STR loci in Han population of Jiangxi province was high. The allelic distribution data at the 6 loci are valuable in population study, individual identification and paternity test for this population.