Inhibitory effect of tanshinone IIA, resveratrol and silibinin on enterovirus 68 production through inhibiting ATM and DNA-PK pathway.

BACKGROUND: Human enterovirus 68 (EV68) is a primary etiological agent for respiratory illnesses, while no effective drug has yet used in clinics largely because the pathogenesis of EV68 is not clear. DNA damage response (DDR) responds to cellular DNA breaks and is also involved in viral replication. Three DDR pathways includes ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK). Natural products proved to be an excellent source for the discovery and isolation of novel antivirals. Among them, tanshinone IIA, resveratrol, silibinin, rutin and quercetin are reported to target DDR, therefore their roles in anti-EV68 are investigated in this study. PURPOSE: This study investigated the anti-EV68 ability of various natural compounds related to DDR. STUDY DESIGN AND METHODS: The methods include cell counting, flow cytometry, western blot, Immunofluorescence staining, comet assays, quantitative real-time RT PCR and short interfering RNAs (siRNAs) for analysis of cell number, cell cycle, protein expression, protein location, DNA damage, mRNA level and knock down target gene, respectively. RESULTS: EV68 infection induced DDR. Down-regulation or inhibition of ATM or DNA-PK lowered DDR in EV68-infected cells and mitigated viral protein expression, however, down-regulation or inhibition of ATR unexpectedly up-regulated DDR, and promoted viral protein expression. Meanwhile tanshinone IIA, resveratrol, and silibinin inhibited ATM and/or DNA-PK activation and decreased viral proliferation, while rutin and quercetin inhibited ATR activation and promoted viral production. The role of them in ATM, DNA-PK and ATR activation was consistent with previous reports. CONCLUSION: Tanshinone IIA, resveratrol and silibinin inhibited EV68 proliferation through inhibiting ATM and/or DNA-PK activation, and they were effective anti-EV68 candidates.

Effect of Renshen Pingfei Decoction, a traditional Chinese prescription, on IPF induced by Bleomycin in rats and regulation of TGF-ß1/Smad3.

AIM OF THE STUDY: Idiopathic pulmonary fibrosis (IPF), one of the clinical common diseases, shares similar pathogenesis with ancient disease "Feibi" in Chinese medicine, Renshen pingfei decoction (RPFS), a classical prescription, was commonly used in treating Feibi. In the current study, the protective role of RPFS in rats model of IPF and the mechanism via regulation of TGF-ß1/Smad3, were evaluated and explored. METHODS: The chemicals of RPFS were analyzed by UPLC-QTOF-MS. Under the optimized chromatographic and MS condition, the major components in RPFS were well separated and detected. An IPF model was established in rats which were induced with Bleomycin (BLM). After treated with corresponding medicine for 7 days, 14 days, 21 days and 28 days respectively, lung function of rats were measured; peripheral blood and bronchoalveolar lavage fluid (BALF) were assessed; histopathological changes and homogenate of lung tissue were detected; TGF-ß1 and Smad3 mRNA and protein expressions in lung tissue were examined as well. RESULTS: 43 signal peaks of chemical components in RPFS were identified by UPLC-QTOF-MS method. Compared with model group, RPFS group exerted significant effects on IPF model rats in improving lung function and decreasing HYP content of lung tissue (P<0.01), reducing the level of TGF-ß1 and NFκB in BALF (P<0.05), decreasing SOD and MDA level in serum (P<0.01), as well as down-regulating TGF-ß1 and Smad3 mRNA and protein expressions of lung tissue (P<0.01). CONCLUSION: RPFS could reduce the lung injury and fibrosis degree and improve lung function of IPF model rats. The protective role might mediated by down-regulating TGF-ß1/Smad3 signaling pathway.

[On the Commonness of San'ao Decoction and Its Analogous Formulas in Facilitating Fei].

San'ao Decoction (SD) and its analogous formulas derived in the following generations are common used prescriptions for treating pulmonary diseases with principal symptoms such as cough and asthma. They are usually compatible with Chinese herbs for facilitating Fei, dispelling wind, resolving phlegm and fluid retention. Material bases in these formulas are mainly derived from Chinese drugs, but dissolution contents of active components are changed and new components are produced after compatibility. By multilevel effect evaluation, these analogous formulas all have commonness in ventilating Fei and superiorities of evidence-based derivation. The effect pathway of commonness was involved in cell structure protection, anti-inflammation, antioxidant, and immunoregulation.

CD4⁺CD25⁺FOXP3⁺ T cells, Foxp3 gene and protein expression contribute to antiasthmatic effects of San'ao decoction in mice model of asthma.

OBJECTIVE: San'ao decoction (SAD) is a commonly used traditional combinatorial formula composed of Herba Ephedrae, Radix Glycyrrhizae and Amygdalus Communis Vas. Early studies showed that in the OVA sensitization asthmatic mice model its compatibility could lower airway reactivity and airway inflammatory cell infiltration. Based on the above results, this study mainly discussed San'ao decoction's immunomodulatory effects on Tregs. METHODS: UPLC-PDA-TOF-MS was applied to analyze chemicals of SAD, and under the optimized chromatographic and MS condition, the major components in SAD were well separated and detected within 22 min. An asthma model was established in BALB/c mice that were sensitized and challenged with ovalbumin. After 2 weeks' treatment, peripheral blood and bronchoalveolar lavage fluid (BALF) were assessed for inflammatory cell counts; histological change of lung tissue were detected; flow cytometry detection of splenic CD4(+)CD25(+)Foxp3(+) Treg cells of the mice were counted; Foxp3 expression in lung tissues were examined as well. RESULTS: 22 Peaks signal chemical components in SAD were identified by UPLC-QTO-MS method. In terms of the percentage of eosinophile in peripheral blood and bronchoalveolar lavage fluid (BALF), SAD groups were significantly lower (p<0.01) than model group. Compared with model group, lung histological changes of SAD groups were reduced; the proportion of CD4(+)CD25(+)Foxp3(+) Treg cells in CD4(+) cells of asthmatic mice also decreased; SAD significantly increased the proportion of CD4(+)CD25(+)Foxp3(+) Treg cells and promoted Foxp3 expression in a mouse model of asthma. CONCLUSION: These results suggest that the antiasthmatic effects of SAD are at least partially associated with CD4(+)CD25(+)Foxp3(+) Treg cells.

Effect of San'ao Decoction on the airway inflammation and hyperresponsiveness in a murine model of lipopolysaccharide-enhanced asthma.

OBJECTIVE: San'ao Decoction (, SAD), as a representative Chinese medicine (CM) formula, was chosen to evaluate the effect of airway inflammation and hyperresponsiveness on the lipopolysaccharide (LPS) enhanced asthma model. METHODS: The asthma model was reproduced in the Balb/C mice sensitized by ovalbumin (OVA), challenged by OVA and LPS. After Balb/C mice's administration of a dose (0.0024 g/kg) of dexamethasone acetate, and three doses (2.2 g/kg, 4.4 g/kg and 8.8 g/kg) of SAD, airway inflammation and responsiveness were observed. The airway inflammation was detected by counting bronchoalveolar lavage fluid (BALF) cells and lung histopathology. Also, differential expressions of interferon-r (IFN-γ), interleukin-4 (IL-4), and IL-5 in the supernatants of BALF were examined. The changes in airway responsiveness indicated by lung resistance (R(L)) and stimulated by acetylcholine (Ach) were determined. RESULTS: Small-dose SAD hardly inhibit airway inflammation or hyperresponsiveness in the LPS-enhanced asthma, while medium-dose and high-dose SAD significantly inhibited the airway hyperresponsiveness, and to some extent, reduced airway inflammation. Meanwhile, the small-dose, medium-dose, and high-dose SAD promoted Th1-type cytokines (IFN-γ) and reduced Th2-type cytokines (IL-4, IL-5) to different extents, which led to a Th1/Th2 balance. CONCLUSION: SAD has a good therapeutic effect on airway hyperresponsiveness in the LPS-enhanced asthma model, but its definite influence on airway inflammation is not remarkable.

Responses in the morphology, physiology and biochemistry of Taxus chinensis var. mairei grown under supplementary UV-B radiation.

The effects of supplemental UV-B radiation on Taxus chinensis var. mairei were studied. Leaf traits, gas exchange parameters and the concentrations of photosynthetic pigments, cellular defense system products, secondary metabolites and ultrastructure were determined. UV-B radiation significantly decreased leaf area (p<0.05). Leaf number, secondary branch number, leaf weight per plant and leaf moisture all increased dramatically (p<0.05). Neither the leaf weight nor the specific leaf weight (SLW) exhibited significant differences between ambient and enhanced UV-B radiation. Gas exchange parameters were all dramatically reduced by enhanced UV-B radiation (p<0.05). The contents of chlorophyll and the chlorophyll a/b ratio were not distinctly affected by UV-B radiation, while carotenoids content significantly decreased (p<0.05). Supplemental UV-B treatment induced significant flavonoid accumulation (p<0.05), which was able to protect plant from radiation damage. Meanwhile, the appendage content, abaxial stomatal density, papilla density and particulate matter content in substomatic chambers increased noticeably by supplemental UV-B radiation, whereas the aperture size of single stomata was diminished. The number and area of plastoglobuli were apparently reduced by UV-B radiation, but stroma and grana lamellae were not destroyed. Our results demonstrated that T. chinensis var. mairei can activate several defense mechanisms against oxidative stress injury caused by supplemental UV-B radiation.

[Effects of San'ao decoction and its analogous prescriptions on airway inflammation in mice with respiratory syncytial virus- and ovalbumin-induced asthma].

OBJECTIVE: To evaluate the effects of San'ao decoction (SAD) and its analogous prescriptions (APs), compounds of traditional Chinese herbal medicine for asthma, on airway inflammation in mice with respiratory syncytial virus (RSV)- and ovalbumin (OVA)-induced asthma. METHODS: A total of 110 mice were randomly divided into control group, untreated group, dexamethasone (DM) group, small-dose SAD (SAD-S) group, large-dose SAD (SAD-L) group, AP I-S group, AP I-L group, AP II-S group, AP II-L group, AP III-S group, and AP III-L group. The asthma model was reproduced by sensitization with multipoint intraperitoneal injection of OVA, followed by repeated inhalation of OVA combined with intranasal instillation of RSV. Cells in bronchoalveolar lavage fluid (BALF) were counted and classified. The supernatant of the BALF was used for detecting the contents of interleukin-4 (IL-4), interleukin-5 (IL-5) and interferon-gamma (IFN-gamma) by enzyme-linked immunosorbent assay. Pathological changes in lung tissue were observed by hematoxylin and eosin staining and the scores of pathological changes were also calculated to determine the degree of inflammation. RESULTS: Compared with the control group, the amounts of lymphocytes, eosinophils, neutrophils in BALF in the untreated group were increased significantly (P<0.01); the changes of lung histopathology in the untreated group were much more serious, and the content of IFN-gamma was sharply decreased, while the contents of IL-4 and IL-5 were significantly increased (P<0.05). The counts of eosinophils in BALF of the treated groups all decreased obviously (P<0.01) as compared with the untreated group. The count of the neutrophils in BALF of the AP II-L group was obviously lower than that in the untreated group (P<0.01). Most of Chinese herbal formulas and DM could increase the level of IFN-gamma, and decrease the level of IL-4. All concentrations of the APs and SAD could decrease the level of IL-5 as compared with the untreated group, especially of the AP II-L and AP I-L (P<0.05, P<0.01). CONCLUSION: SAD and its APs had some therapeutic effects on RSV-induced asthma in mice. Among the formulas, AP II has a better therapeutic efficacy in treatment of asthma by decreasing the amount of neutrophils.

P53-mediated cell cycle arrest and apoptosis through a caspase-3- independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells.

AIM: To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro. METHODS: The viability of oridonin-treated MCF-7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein (Hsp)90, p53, p-p53, p21, Poly (ADP-ribose) polymerase (PARP), and the inhibitor of caspase-activated DNase (ICAD) protein expressions were detected by Western blot analysis. RESULTS: Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z-VAD-fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of DeltaPhimit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by caspase-3. CONCLUSION: DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.