The therapeutic effect of Loranthus parasiticus lignan derivatives on collagen-induced arthritis in rats through the SHBG/NFκB pathway.

Lignan-rich beans, nuts, and various seeds are the main foods with antioxidative and hormone-modulating activities. Although the role of lignans in mediating hormone-dependent cancers and cardiovascular diseases is well characterized, the function of lignans in anti-arthritic activity and its underlying mechanisms remain unknown. Three new lignan derivatives, (-)-nortrachelogenin, trachelogenin, and matairesinol, were extracted from Loranthus parasiticus. After establishing the collagen-induced arthritis (CIA) model by intradermal injection of collagen, rats were treated with three new lignan derivatives ((-)-nortrachelogenin: 37%; trachelogenin: 27%; matairesinol: 25.7%) at a concentration of 50 mg/kg and 100 mg/kg, or methotrexate at 0.3 mg/kg. Mixed lignan derivatives significantly attenuated the immune responses in the joints of CIA rats, leading to lower levels of proinflammatory cytokines (IL-6 and TNF-α) and higher levels of free androgen in the serum compared to the CIA model. The results of molecular docking using AutoDock Vina showed that the lignan derivative (-)-nortrachelogenin was the most effective compound for binding to sex hormone-binding globulin (SHBG), thus inhibiting the activity of NFκB in LPS-stimulated macrophages. In this study, (-)-nortrachelogenin was identified as a novel natural lignan derivative with previously unrecognized anti-inflammatory activity. Its molecular mechanism appears related to the regulation of the NFκB/SHBG pathway. Our findings suggest that further application of sex hormone-like compounds in the treatment of rheumatoid arthritis and the potential clinical applications of (-)-nortrachelogenin are promising.

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.

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.

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.

Construction of a ceRNA Network Related to Rheumatoid Arthritis.

(1) Background: Rheumatoid arthritis (RA) is a common systemic autoimmune disease affecting many people and has an unclear and complicated physiological mechanism. The competing endogenous RNA (ceRNA) network plays an essential role in the development and occurrence of various human physiological processes. This study aimed to construct a ceRNA network related to RA. (2) Methods: We explored the GEO database for peripheral blood mononuclear cell (PBMC) samples and then analyzed the RNA of 52 samples (without treatment) to obtain lncRNAs (DELs), miRNAs (DEMs), and mRNAs (DEGs), which can be differentially expressed with statistical significance in the progression of RA. Next, a ceRNA network was constructed, based on the DELs, DEMs, and DEGs. At the same time, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis were used to validate the possible function of the ceRNA network. (3) Results: Through our analysis, 389 DELs, 247 DEMs, and 1081 DEGs were screened. After this, a ceRNA network was constructed for further statistical comparisons, including 16 lncRNAs, 1 miRNA, and 15 mRNAs. According to the GO and KEGG analysis, the ceRNA network was mainly enriched in the mTOR pathway, the dopaminergic system, and the Wnt signaling pathway. (4) Conclusions: The novel ceRNA network related to RA that we constructed offers novel insights into and targets for the underlying molecular mechanisms of the mTOR pathway, the dopaminergic system, and the Wnt signaling pathway (both classic and nonclassic pathways) that affect the level of the genetic regulator, which might offer novel ways to treat RA.

Studies on separating the impedance change components of blood vessels and ventricles in thorax from mixed impedance signals on chest surface.

PURPOSE: The aim of the present study is to separate the impedance change components of the blood vessels and ventricles in thorax from the mixed impedance signals detected on the chest surface. METHODS: The mixed impedance signals on the chest surface are measured with a 15 electrode lead system. The thoracic impedance equations are established and solved iteratively with the algebraic reconstructed technique. Experiments were performed on 80 healthy, otherwise normal, adults. RESULTS: Five impedance change components for the aorta (AO), blood vessel in left lung (PL), blood vessel in right lung (PR), left ventricle (LV), and right ventricle (RV) are separated from the mixed impedance signals. The experiments show that the main waveform of the ventricular components LV and RV is contrary to that of the vascular components AO, PL, and PR, and the negative peak point of the waveform graphs of LV and RV are in phase with the second cardiac sound (S2). The waveform graphs of various components correspond with the physiological activities of the heart and blood vessels in a cardiac cycle. The statistical results for 80 normal adults show that the amplitude of AO is the largest and that of PL and PR is the next, while that of LV and RV is the smallest. There are significant differences between them (P < 0.01). CONCLUSIONS: The mathematical model and the measurement method for the separation in the present paper are feasible.

Tumor-Targeted Gene Silencing IDO Synergizes PTT-Induced Apoptosis and Enhances Anti-tumor Immunity.

Background: Photothermal therapy (PTT) has been demonstrated to be a promising cancer treatment approach because it can be modulated to induce apoptosis instead of necrosis via adjusting irradiation conditions. Recently, an abscopal anti-tumor immunity has been highlighted, in which PTT on the primary tumor also induced repression of distant tumors. In PTT cancer treatments, the mechanism and the role of immune checkpoints to enhance anti-tumor immunity needs to be investigated. Methods: We prepared a multi-functional gold nanorod reagent, GMPF-siIDO, that is composed of gold nanorods (GNRs) that act as the nano-platform and photothermal sensitizer; folic acid (FA) as the tumor-targeting moiety; and IDO-specific RNA (siIDO) as an immune-stimulator functionality for inducing anti-tumor immunity. For this study, we adjusted the irradiation condition of PTT to induce apoptosis and to silence the immune checkpoint indoleamine 2,3 dioxygeonase (IDO), simultaneously. Results: Our studies provide evidence that photothermal effects kill tumor cells mainly via inducing apoptosis, which can significantly improve antitumor immunity when IDO was down-regulated in TME through significant increases of localized CD8+ and CD4+ lymphocytes in tumor tissue, the downregulation of CD8+ and CD4+ lymphocyte apoptosis, and the upregulation of antitumor cytokines, TNF-α and IFN-γ. Conclusion: In this study, we, for the first time, validated the role of IDO as a negative regulator for both PTT-induced tumor cell apoptosis and anti-tumor immunity; IDO is a critical immune checkpoint that impedes PTT while combination of gene knockdown of IDO in TME enhances anti-tumor efficacy of PTT.

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.].

Curcumin attenuates collagen-induced rat arthritis via anti-inflammatory and apoptotic effects.

Curcumin is a natural herbal product that has been popularly used to treat autoimmune diseases in China; however, its effects on rheumatoid arthritis and its mechanism are not clear. The main purposes of this study are to explore the therapeutic effects of curcumin on collagen-induced arthritis (CIA) rats and the pharmacological mechanism. In the present study, CIA rats were established by injecting bovine type II collagen. Curcumin and methotrexate were then orally administered daily, and the swelling degree of the hind limb joints was scored every two days. Histopathological changes were observed by hematoxylin-eosin staining. The levels of cytokines (TNF-α, IL-1ß, IL-17 and TGF-ß) were detected by radioimmunoassay, while the expression of IκBα and COX-2 was detected by Western blot. In addition, cell viability was detected by CCK-8 assay, and the effect of curcumin on macrophage apoptosis was detected by flow cytometry and TUNEL assay. The results indicated that in vivo curcumin attenuated the degree of joint swelling of rats and the further development of joint histopathology. Moreover, it downregulated the levels of cytokines. In vitro curcumin inhibited the degradation of IκBα and reduced the production of COX-2 in LPS-induced inflammatory RAW264.7 cells. Importantly, curcumin significantly induced macrophage apoptosis. In conclusion, in this study, we have demonstrated that curcumin exerts therapeutic effects on arthritis in CIA rats and has a strong pharmacological activity on reducing the inflammatory response in macrophages. Its mechanism may be related to the inhibition of the NF-κB signaling pathway and the promotion of macrophage apoptosis.

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.

Comparing square root method of measuring the cardiac output by means of aortic impedance change component to Kubicek's method.

PURPOSE: The aim of this study is to explore a calculated method used to measure the cardiac output using the aortic impedance change component of reconstructed impedance cardiography. METHODS: Routine impedance cardiography was measured using Kubicek's method with four ring electrodes. The thoracic mixed impedance changes were measured by six leads, which consisted of 15 electrodes. The aortic impedance change component was separated from six thoracic mixed impedance changes through waveform reconstruction. The square root formula used to calculate the cardiac output was deduced based on the thoracic impedance change equation and the aortic volume change hypothesis during the systole period. The cardiac outputs of 180 normal adults and 72 patients with cardiac insufficiency who could still walk freely were contrastively computed with both Kubicek's formula and the square root formula. RESULTS: For 180 normal adults, the cardiac index (CI) computed with the square root formula was 3.60 ± 0.45 L/min/m2 , with normal values ranging from 2.7 to 4.5 L/min/m2 . A total of 163 cases (90.6%) had a CI in the standard range (2.7-4.3 L/min/m2 ) adopted in clinical applications. The CI computed with Kubicek's formula was 3.61 ± 0.86 L/min/m2 , with normal values ranging from 1.9 to 5.3 L/min/m2 , and only 115 cases (63.9%) had a CI in the above standard range. Among the 72 patients with cardiac insufficiency, 20 (27.8%) patients had a CI < 2.0 L/min/m2 with Kubicek's formula. Of these 20 cases, 9 cases had a CI < 1.5 L/min/m2 , and 4 cases had a CI < 1.1 L/min/m2 . In contrast, none of the 72 patients had a CI < 2.0 L/min/m2 with the square root formula. In addition, the influence of the chest circumference on the CI was lower for the square root formula than for Kubicek's formula. CONCLUSIONS: The CI calculated with the square root formula had a better normal value range, was more accurate for the patients with cardiac insufficiency, and was less affected by the chest circumference than the CI calculated with Kubicek's formula.

Analyzing the formation of normal and abnormal O waves in thoracic impedance graph using the impedance change components for aorta, blood vessels in lung and ventricles.

BACKGROUND: Many measurements of thoracic impedance graph show that the small C wave and big O wave appear often for patients with cardiac insufficiency, and the O/C ratio is bigger. And for the normal body, especially a younger one, the bigger O wave may also appear. But since the amplitude of the C wave of a normal body is bigger, the O/C ratio is smaller. The aim of the present paper is to investigate the formation mechanism of the normal and abnormal O waves in thoracic impedance graph. METHODS AND RESULTS: The thoracic mixed impedance changes are measured with 6 leads consisting of 15 electrodes. The impedance change components for the aorta (AO), blood vessel in left lung (PL), blood vessel in right lung (PR), left ventricle (LV) and right ventricle (RV) are separated from thoracic mixed impedance changes by means of establishing and solving the thoracic impedance equations. The amplitudes of the O and C waves of various impedance change components are measured for 50 normal healthy adults and 34 patients with cardiac insufficiency. The formation mechanism of normal and abnormal O waves in thoracic impedance graph is analyzed using the superposition of the O waves of the above impedance change components. Detection subjects are 50 healthy adults and 34 hospital patients with cardiac insufficiency. (1) Thoracic impedance graph: The O/C ratios of the normal group are significantly smaller than that of the abnormal group, p < 0.001. The O wave of first lead (E1-E1') is significantly bigger than that of leads 4 and 5 (E4-E4' and E5-E5') in the normal group, p < 0.001. (2) The impedance change component: The O waves of the AO, PL, and PR are significantly smaller than that of the LV and RV in the normal group, p < 0.001. The O wave and O/C of the AO, PL and PR of normal group are significantly smaller than that of the abnormal group, p < 0.001. CONCLUSIONS: The O wave of the thoracic impedance graph is formed due to the superposition of the O waves of the impedance change components for the aorta, blood vessels in lung and ventricles.

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.

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.

Mechanism of the formation for thoracic impedance change.

The purpose of this study is to investigate the mechanism of the formation for thoracic impedance change. On the basis of Ohm's law and the electrical field distribution in the cylindrical volume conductor, the formula about the thoracic impedance change are deduced, and they are demonstrated with the model experiment. The results indicate that the thoracic impedance change caused by single blood vessel is directly proportional to the ratio of the impedance change to the basal impedance of the blood vessel itself, to the length of the blood vessel appearing between the current electrodes, and to the basal impedance between two detective electrodes on the chest surface, while it is inversely proportional to the distance between the blood vessel and the line joining two detective electrodes. The thoracic impedance change caused by multiple blood vessels together is equal to the algebraic addition of all thoracic impedance changes resulting from the individual blood vessels. That is, the impedance changes obey the principle of adding scalars in the measurement of the electrical impedance graph. The present study can offer the theoretical basis for the waveform reconstruction of Impedance cardiography (ICG).