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As a classic prescription, Wuji Pills is composed of Coptidis Rhizoma, Euodiae Fructus Preparata, and stir-fried Paeo-niae Radix Alba at the ratio of 6∶1∶6. The practical application of it is limited compared with other famous Chinese medicine prescriptions. Only one company produces Wuji Pills in China. In this study, ultra-performance liquid chromatography quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS/MS) was used to analyze and identify 26 identical compounds from Wuji Pills and drug-containing plasma of rats. Based on these components, 46 potential targets were screened out with network pharmacology methods, followed by the component-target network construction, Gene Ontology(GO) term enrichment, Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and disease prediction. It was concluded that Wuji Pills acted on core targets such as PTGS2, PTSG1, NCOA2, HSP9 OAD1, and RXRA through magnoflorine, hydroxyevodiamine, daucosterol, and berberine and exerted pharmacodynamic effects through various pathways such as calcium ion signaling pathway, phosphatidylinositol-3-kinase-protein kinase B(PI3 K-Akt) signaling pathway, and vascular endothelial growth factor(VEGF) signaling pathway. Thus, Wuji Pills has therapeutic potential for Alzheimer's disease, diabetes mellitus, myocardial ischemia, and other diseases in addition to the conventional disease(irritable bowel syndrome, IBS). The above research results can provide a reference for the comprehensive interpretation of the pharmacodynamic basis of Wuji Pills and the expansion of clinical application. At the same time, a lot of components in serum and the in vivo transformed and metabolized components of Wuji Pills have similar structure and relative molecular weight. In theory, these components may show additive effects and the competitive/antagonistic effects on the same target. According to the hypothesis ofquot;additive effect of multiple components for a single targetquot; in traditional Chinese medicine, multiple similar components may exert the additive effects on local targets. This study can partly prove the scientificity of this hypothesis and provide laboratory evidence.

Somatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency.This study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs.The viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. TheMost sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. TheSow urine-derived cells could be used to produce SCNT embryos.

Excitatory toxicity(ET) is an important factor of neuropathic pain(NPP) induced by central sensitization(CS), and the association of pannexin-1(Panx1)-Src-N-methyl-D-aspartate receptor subunit 2 B(NMDAR-2 B) is an important new pathway for ET to initiate CS. The present study confirmed whether the central analgesic effect of Chuanxiong Rhizoma extract(CRE) was achieved through the synchronous regulation of the brain and spinal pathways of Panx1-Src-NMDAR-2 B. In this study, dynamic and simulta-neo-us microdialysis of the brain and spinal cord in vivo combined with behavioristics, high performance liquid chromatography(HPLC)-fluorescence detection, microdialysis analysis(ISCUS~(flex)), ultrasensitive multifactorial electrochemiluminescence immunoassay, ELISA, and Western blot was employed to investigate the protein expression of NMDAR-2 B, Src, and Panx1, extracellular excitatory amino acids, cytokines, energy metabolites, and substance P in spinal dorsal horn(SDH) and anterior cingulate cortex(ACC) after CRE intervention with the rat model of spared sciatic nerve injury(SNI) as the experimental tool. Compared with the sham group, the SNI group exhibited diminished mechanical withdrawal threshold(MWT)(Plt;0.01), increased cold spray scores(Plt;0.01), glutamate(Glu), D-serine(D-Ser), and glycine(Gly) in extracellular fluids of ACC, and Glu, D-Ser, interleukin-1β(IL-1β), and lactic acid(Lac) in extracellular fluids of SDH(Plt;0.05), dwindled tumor necrosis factor(TNF-α)(Plt;0.05), and elevated protein levels of NMDAR-2 B, Src, and Panx1 in ACC(Plt;0.05). Compared with the SNI model rats, high-and medium-dose CRE(CRE-H/M) could potentiate the analgesic activity as revealed by the MWT test(Plt;0.05) and CRE-M enabled the decrease in cold spray scores(Plt;0.05). CRE-H/M could inhibit the levels of Glu, D-Ser and Gly in the extracellular fluids of ACC(Plt;0.05), and the levels of Glu in the extracellular fluids of SDH(Plt;0.05) in SNI rats. CRE-M significantly increased the levels of glucose(Gluc), Lac, interferon-gamma(IFN-γ), keratinocyte chemoattractant/human growth-regulated oncogenes(KC/GRO), and IL-4 in extracellular fluids of SDH in SNI rats(Plt;0.05). CRE-H/M/L could also inhibit the levels of NMDAR-2 B, Src and Panx1 in ACC and SDH in SNI rats(Plt;0.05). The central analgesic effect of CRE is presumedly related to the inhibited release of excitatory amino acid transmitters(Glu, D-Ser and Gly) in ACC and SDH of SNI rats, decreased protein expression of NMDAR-2 B, Src and Panx1 in the two regions, and the regulation of the Panx1-Src-NMDAR-2 B pathway in the spinal cord and brain. The above findings partially clarified the scientific basis of clinical analgesic effect of Chuanxiong Rhizoma.

This study aimed to investigate the effect and the possible mechanism of Shenlian( SL) extract on tumor necrosis factor-α( TNF-α)-induced ECV304 injury. After the establishment of TNF-α-induced ECV304 cells injure model,MTT assay was used to detect cell viability and the level of reactive oxygen species( ROS) was measured by flow cytometry. The contents of superoxide dismutase( SOD),malondialdehyde( MDA),nitric oxide( NO),endothelin-1( ET-1) and interleukin-1β( IL-1β) in the supernatant were detected by biochemical method and enzyme linked immunosorbent assay( ELISA). The expression levels of apoptosis-related proteins B-lymphoma-2 gene( Bcl-2),Bcl-2 associated X protein( Bax),caspase-3,caspase-9 and nuclear factor E2 associated factor2( Nrf2)/Kelch like epichlorohydrin associated protein-1( Keap1) signaling pathway related proteins Nrf2,Keap1,quinone oxidoreductase( NQO1) and heme oxygenase 1( HO-1) were detected by Western blot. The results showed that 50 μg·L-1 TNF-α significantly damaged ECV304 cells,induced the impairment of cell viability( Plt;0. 01),the increase of ROS production,the decrease of SOD activity,and the increase of MDA,NO,ET-1 and IL-1β( Plt;0. 01),meanwhile,it caused the up-regulation of Keap1,caspase-9 and Bax protein expression,and down-regulation of NQO1 and Bcl-2 protein expression( Plt;0. 05) compared with the control group.Compared with the model group,SL extract reduced the damage of ECV304 cells induced by TNF-α,improved cell viability,reduced ROS production,increased SOD activity and decreased MDA,NO,ET-1,IL-1β content( Plt;0. 01 or Plt;0. 05). In addition,SL extract also down-regulated the protein expression levels of Keap1,caspase-3,caspase-9 and Bax,and increased the protein expressions of Nrf2,NQO1,HO-1 and Bcl-2( Plt;0. 01 or Plt;0. 05). The above results indicate that SL extract can provide protective effect on ECV304 cells injury induced by TNF-α,alleviate oxidative stress injury,inflammation and apoptosis,and its mechanism may be related to regulating Nrf2/Keap1 signaling pathway.

Shenlian extract (SL), extracted from Salvia miltiorrhiza Bunge and Andrographis paniculata (Burm. f.) Nees, has been proved to be effective in the prevention and treatment of atherosclerosis. Recently, we have partially elucidated the mechanisms involved in the therapeutic effects of SL on myocardial ischemia (MI). However, the underlying mechanisms remain largely unclear.This study aims to explore the potential molecular mechanism of SL on MI on the basis of network pharmacology.First, the main active ingredients of SL were screened in the Traditional Chinese Medicine Integrated Database, and the MI-associated targets were collected from the DisGeNET database. Then, we used compound-target and target-pathway networks to uncover the therapeutic mechanisms of SL. On the basis of network pharmacology analysis results, we assessed the effects of SL in MI rat model and oxygen glucose deprivation model of H9c2 cells and validated the possible molecular mechanisms of SL on myocardial injury in vivo and in vitro.The network pharmacology results showed that 37 potential targets were recognized, including TNF-α, Bcl-2, STAT3, PI3K and MMP2. These results revealed that the possible targets of SL were involved in the regulation of inflammation and apoptosis signaling pathway. Then, in vivo experiments indicated that SL significantly reduced the myocardial infarction size of MI rats. Serum CK-MB, cTnT, CK, LDH, and AST levels were significantly decreased by SL (P 0.05 or P 0.01). In vitro, SL significantly increased H9c2 cell viability. The levels of inflammation factors including TNF-α and MMP2 were significantly decreased by SL (P 0.05 or P 0.01). TUNEL and Annexin V/propidium iodide assays indicated that SL could significantly decrease the cell apoptotic rate in vivo and in vitro (P 0.05 or P 0.01). The remarkable upregulation of anti-apoptotic Bcl-2 and downregulation of pro-apoptotic Bax protein level further confirmed this result. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the PI3K-AKT and JAK2-STAT3 pathways were significantly enriched in SL. Compared with the model group, SL treatment significantly activated the PI3K-AKT and JAK2-STAT3 pathways in vivo and in vitro according to Western blot analyses.SL could protect the myocardium from MI injury. The underlying mechanism may be related to the reduction of inflammation and apoptosis by activating the PI3K/AKT and JAK2/STAT3 pathways.

Background: In the freezing process of boar sperm, there are obvious differences of freezability between individuals. Studies suggest that specific freezability markers might be useful in good (GFE) and poor freezabitity ejaculates (PFE) selection prior to cryopreservation. Some potential markers of boar sperm freezability have been found from spermatozoa, little attention has been paid to seminal plasma. The seminal plasma is composed of secretions from testis, epididymis, and accessory sex glands, and the exposure of spermatozoa to small molecules such as metabolites can affect the sperm functions. However, details and significance of the seminal plasma metabolome related to boar sperm freezability are unknown. Therefore, the main aim of this study was to explore the difference in the metabolic level of seminal plasma between boars with differential freezability, and to explore the biomarkers of semen freezing tolerance. Results: A total of 953 metabolites were identified in boar semen plasma by UHPLC-qTOF-MS analysis, and 50 metabolites show significant change between GFE group and PFE group. Further, twelve metabolites were subjected to metabolic target analysis and three metabolites (D-Aspartic acid, N-Acetyl-L-glutamate (NAG), and Inosine) show differences.Conclusions: There is significant differece on metabolome of seminal plasma between GFE and PFE individuals. The D-Aspartic acid, NAG, and Inosine in seminal plasma may be potential markers for assessing sperm cryopreservation resistance in boars.

The aim of this paper was to obtain low toxicity and high efficiency anti-tumor Chinese medicine through screening the combination ratios of Momordicae Semen and Epimedii Folium, and to explore the anti-tumor mechanism of the combination of two drugs by observing their effect on apoptosis-related proteins in cancer cells. Methyl thiazolyl tetrazolium(MTT) assay was used to observe the effect of drug combination on the proliferation of tumor cells from different tissue sources. The effects of the combination of the two drugs on tumor cells were analyzed by Compusyn software. Plate cloning assay was used to observe the effect of combination of these two drugs on the proliferation of A549 cells in vitro. The expression of reactive oxygen species(ROS) and apoptotic proteins p53, Bcl-2 and Bax were compared by using ROS kit and Western blot. Lewis lung cancer model was used to observe the anti-tumor effect of drugs in vivo. The results showed that the anti-tumor effect of their ethanol extract was more significant than that of water extract, and the anti-proliferation effect was strongest when the ratio was 1∶1(Plt;0.05). Compusyn analysis showed that the combination of the two drugs had synergistic effect. Further studies showed that after combined use, the number of clonogen formation in A549 cells was significantly reduced(Plt;0.01); ROS production was increased; the expression of apoptosis-related protein p53 was up-regulated, and the ratio of Bcl-2/Bax was decreased. In vivo animal study showed that the tumor inhibition rate was 53.06%(Plt;0.05) in the high dose group. As compared with the single use of the two drugs, the combination of the two drugs had more significant anti-proliferative effect on tumors, and the optimum ratio was 1∶1. The combination of the two drugs at a ratio of 1∶1 inhibited the proliferation of various tumor cells, and had no significant effect on normal liver cells LO2 when compared with other ratios. Therefore, it can be preliminarily inferred that the combination of the two drugs may have the effect of synergism and detoxification. Further studies showed that the combination of the two drugs can significantly inhibit the proliferation of A549 cells, and its mechanism may be related to the activation of endogenous apoptotic pathway. In vivo experiments also showed that the tumor inhibition rate increased with the increase of drug concentration.

Artemisinin was isolated from traditional Chinese herb Artemisia annua for treating malaria. A series of derivatives,like dihydroartemisinin,artesunate,artemether,artether,had the same core chemical structure,and sesquiterpene lactone containing peroxide bridge constitute the basic chemical structure. Besides anti-malaria,artemisinin family drugs were found to ameliorate many different diseases,which have attracted wide attention in recent years. Among different diseases,artemisinin family drugs were found to have T lymphocytes immunomodulation effects,including activation,proliferation,differentiation,apoptosis and subsets function. Because T cell immunologic response is the key point of many diseases,and impact the pathogenic process,therapeutic effect and prognosis,the drug studies with it as the target have become hotspots in recent years. Studies of artemisinin family drug on T cell immunomodulation were still at the initial stage and involved in different disease; furthermore,T cell immune process involves complicated molecular mechanism,it is imperative to summarize the advance of current studies for further systematic explanation and exploration of their characteristics and mechanisms. This article will summarize the research progress of artemisinin family drugs for malaria,autoimmune disease,hypersensitivity reaction,tumor,schistosomiasis and AIDS relating to T cell immune modulation,so as to provide basic and professional reference for related research and application.

Metastasis of breast cancer is the vital step for malignant progression. During such a process, hematogenous metastasis is an indispensable approach for the dissemination of cancer cells. A platelet, contributes to hypercoagulable state, and is also identified the crucial factor in the coagulation system for supporting metastasis. Therefore, the relationship of a platelet and a tumor cell plays a critical role in tumor cell metastasis. Consequently, inhibiting tumor cell‑induced platelet aggregation (TCIPA) is recongnized as a crucial target on suppression of tumor metastasis such as aspirin (ASA). Under such circumstance, here we report that, through dissociating the tumor‑platelet (T‑P) complex, 80% ethanol extracts of Caulis Spatholobi (SET) successfully alleviated the hypercoagulation state, thereby reducing tumor metastasis and improving the prospects of survival in breast cancer cell model. Through MTT and anti‑aggregation assay stimulated by ADP, we detected the optimum treatment time and the optimum dose of SET. By using confocal microscopy, we observed that SET can strongly block the formation of T‑P complex in vitro. The result was further quantified and confirmed by the FACS analysis. The fluorescent value of T‑P complex was obviously decreased in the drug‑treated groups. In vivo, 4T1 cells were injected through the mouse tail vein for dynamic visualization by small animal imaging system. The metastatic intensity was quantified and the survival curve was analyzed. Additionally, general observation and hematoxylin and eosin (H&E) staining of lung tissue was performed. SET exerted an obvious effect on the inhibition of metastasis and increasing the survival rate of mice. For the molecular mechanism study of anti‑TCIPA, zymography and RT‑PCR assay preliminarily revealed the molecular mechanism of SET in the regulation of P‑T interaction. Collectively, through drug efficacy identification and pharmacological revealing, we have obtained a promising candidate for the interference of breast metastasis by suppressing TCIPA, which will be beneficial for clinical cancer treatment.

This paper was mainly to discuss the potential role and mechanism of Lianhua Qingwen Capsules(LHQW) in inhibiting pathological inflammation in the model of acute lung injury caused by bacterial infection. For in vitro study, the mRNA expression of MCP-1 in RAW264.7 cells and THP-1 cells, the content of MCP-1 in cell supernatant, as well as the effect of LHQW on chemotaxis of macrophages were detected. For in vivo study, mice were randomly divided into 7 groups, including normal group, model group(LPS 5 mg·kg~(-1)), LHQW 300, 600 and 1 200 mg·kg~(-1)(low, middle and high dose) groups, dexamethasone 5 mg·kg~(-1) group and penicillin-streptomycin group. Then, the anal temperature was detected two hours later. Dry weight and wet weight of lung tissues in mice were determined; TNF-α and MCP-1 levels in alveolar lavage fluid and MCP-1 in serum were detected. In addition, the infiltration of alveolar macrophages was also observed and the infiltration count of alveolar macrophages was measured by CCK-8 method. HE staining was also used to observe the inflammatory infiltration of lung tissues in mice. Both of the in vitro and in vivo data consistently have confirmed that: by down-regulating the expression of MCP-1, LHWQ could efficiently decrease the chemotaxis of monocytes toward the pulmonary infection foci, thus blocking the disease development in ALI animal model.

Dihydroartemisinin (DHA) is a derivative of the herb Artemisia annua L. that has prominent immunomodulatory activity, however, its underlying mechanism remains elusive. Inflammatory bowel disease (IBD) is an idiopathic inflammatory condition characterized as an autoimmune disorder that includes dysfunctions in the T helper (Th)/T regulatory cell (Treg) balance, which normally plays pivotal roles in immune homeostasis. The aim of this study was to explore the potential of DHA to ameliorate IBD by restoring the Th/Treg cell balance. To this end, we established mouse models of colitis induced by oxazolone (OXA) and 2,4,6-trinitro-benzene sulfonic acid (TNBS). We then treated mice with DHA at 4, 8, or 16 mg/kg/day. DHA treatment ameliorated colitis signs and reduced lymphocyte infiltration and tissue fibrosis. Moreover, DHA decreased the numbers of Th1 and Th17 cells and Th9 and Th22 cells in TNBS- or OXA-induced colitis, respectively, and increased Tregs in both models. DHA (0.8 mg/mL) also inhibited activated CD4+ T lymphocytes, which was accompanied by apoptosis induction. Moreover, it promoted heme oxygenase-1 (HO-1) production in vitro and in vivo, concomitant with CD4+ T cell apoptosis and restoration of the Th/Treg balance, and these effects were blocked by treatment with the HO-1 inhibitor Sn-protoporphyrin IX. Overall, these results suggest that DHA is a novel and valuable candidate for IBD therapy or Th/Treg immunoregulation.

In the background of the high incidence and high mortality of cardiovascular diseases,atherosclerosis is the main pathological feature of cardiovascular diseases and the core pathological basis for disease progression. In the evolution of atherosclerotic plaques,the rupture of unstable plaques,plaque shedding and formation of thrombosis are the most dangerous parts. In this process,the formation of plaque fibrosis is the core mechanism regulating plaque stability. Additionally,fibrosis reflects dynamic changes in the inflammatory processes and pathological changes. In view of the inflammation regulation and fibrosis regulation,this paper clarified the process of atherosclerotic plaque,explained the roles of relevant inflammatory cells and cytokines in plaque stability,and summed up drug researches related with stable plaque in recent years. In the future,improving the fibrosis will be a new idea for stabilizing plaque in atherosclerosis drug development.

Human health has been severely threatened by malignant tumors continuously.Rational and effective drug use provides an effective means for the treatment of malignant tumors,and is expected to become an important way to solve the problem of tumor treatment in the future.In recent years,with the escalation of new cancer theories and the emergence of clinical drug resistance,innovative research and development of anti-cancer drugs has always been a hot spot and focus in cancer research.Among them,the discovery of novel anti-cancer drugs from natural compound is of top priority due to its strong anti-cancer efficacy and the abundant drug resources.Therefore,it is imperative to systematically summarize the cutting-edge advancements of the natural products and their potential pharmacological mechanisms according to the characteristics of tumor progression,and put forward the new directions and trends for further development of anti-cancer natural products in the future.Specifically,the research advancements on anti-cancer effect of natural products were reviewed,focusing on both the traditional and innovative application.We hope this review could bring the light on the research path of the natural anti-cancer products clearly and comprehensively,and also provide inspirations for innovative,safer and more effective anti-cancer drug development and exploration.

This study was designed to investigate the activity of Shenlian tablet in stabilization of the atherosclerosis (As) plaque in apoE(-/-) mice and explore the mechanisms. Rat peritoneal mast cells were randomly allocated and treated with Shenlian tablet (100, 50, 25, 12.5 mg·L(-1)) or cromoglicate sodium (200 μg·L(-1)) for 2 h before exposure to substance P. Histamine, tryptase, IL-1β and NF-κB were measured in the cell culture supernatant by ELISA assay. The plaque formation was induced by common carotid artery cannula method combined with high-fat diet in apoE(-/-) mice, and the plaque instability was induced by substance P through local mast cell degranulation. Mice were divided into eight groups that included the model 1 (M1, sham-operated group), M2 (carotid artery cannula combined with high-fat diet）, M3 (M2 combined with substance P 0.5 μg/mouse, Shenlian extract (95, 190 and 380 mg·kg(-1)·d(-1)), atorvastatin (2.6 mg·kg-1·d(-1)) and normal control group. Total cholesterol (TC), high-density lipoprotein (HDL-C), high-sensitivity C-reactive protein (hs CRP), matrix metalloproteinases 9 (MMP-9) and histamine were measured by ELISA. Thickness, plaque area, mast cell degranulation were observed by hematoxylin and eosin staining, toluidine blue staining. CD117 antigen expression were observed by confocal microscopy. Intracellular phosphorylation was detected using the Bio-Plex 6-plex phosphoprotein assay kit. The results show that the mast cell membrane was stabilized by Shenlian tablet. Histamine, tryptase, interleukin l-β and NF-κB exhibited a significantly reduction in the Shenlian tablet-treated group (P0.05 or P0.01). Substance P significantly enhanced activation and degranulation of adventitial mast cells. In addition, it increased adventitia inflammatory cells infiltration and promoted intraplaque hemorrhages in apoE(-/-) mice model group. The proliferation, degranulation and inflammation of mast cell were significantly inhibited by Shenlian tablet. On the other hand, the same treatment decreased hs-CRP, MMP-9 and histamine in serum. IκB, p38 MAPK phosphorylation, intraplaque hemorrhage and collagen degradation were reduced in the presence of Shenlian tablet, which increased the stability of the As plaque. The results show that the vulnerable plaque model induced by mast cell activation in adventitia was established. Shenlian tablet exhibited a protective effect in this model. Shenlian tablet may increase the plaque stability via inhibition of mast cell-mediated inflammatory response.

This article proposes a new thought on the study of "main effect" of traditional Chinese medicine (TCM) formulae. The blood concentrations of the pharmacodynamic substances of Chinese material medica（CMM）are usually very low, with lower toxic and side effects than western medicine. Therefore, according to a recent hypothesis of additive effect of multiple components for a single target, local targets in multi-component multi-target synergistic effect network of TCM may have the additive effect of similar components. Studies on the disposition of CMM showed that a constituent could bebio-transformed to many metabolites; these compounds with a similar structure are likely to have the same pharmacological effects on the same target, which could provide experimental evidences for the hypothesis of "additive effect". The authors of this article further believe that additive effect of TCM multi-components only comes up under a limited conditions/concentration. Because of the complexity of TCM-organism system, the complex effect of multicomponent addition and competition/antagonism is more likely to appear in single targets of drug effect. This complex effect may be the key to impact the synergistic effect of TCM multi-targets. In theory, choose and create a single target additive effect could realize the scientific compatibility of TCM and improve the curative effect and attenuate toxicity. According to the clinical demand and under the guidance of the above thought, we proposed the "main effect" of TCM formulae. Because traditional Chinese medicine (compounds) have diverse and complex effects, how to better study TCM formulae compatibility mechanism and improve the curative effect? Efforts shall be made to select one or several effects relating to clinical specific syndromes from the complex and diverse effects of TCM as the "main effect". The "main effect" of TCM formulae is the macroscopic manifestation of the synergistic effect of multi-component/multi-target. The study of the Formulae "main effect" can contain at least two aspects: one is the study of pharmacokinetic application of TCM formulae, and another is the study for pharmacodynamics effect. In the study of main effect, there are two main elements. First, which drug targets are directly related to the main effect? This requires identifying the target network. Second, which drug components positively or negatively control the single target of the target network? And what change in single target effect as well as the multi-target synergistic effect will be caused by the regulatory component concentration or the change in number? These two elements is the key to elucidate the mechanism of compound action and compatibility mechanism of Chinese herbal compound formulae. Through the study of the main effect, the clinical curative effect and the mechanism of the TCM formulae shall be improved.

It is important to utilize an antioxidant to ameliorate oxidative damage during boar sperm cryopreservation and thawing. Some studies have shown that Astragalus polysaccharide (APS) has antioxidant capabilities in sperm storage at low temperatures. However, the effects of APS on thawed sperm are unclear. In this study, the effect of supplementation of thawing boar semen extender with APS (0.5, 1, 5, 10 mg/mL) on sperm quality parameters (viability, motility, acrosome integrity and mitochondrial activity) was evaluated. Next, we investigated the effect of APS (0.5 mg/mL) supplementation on antioxidant parameters. Semen from two straws was thawed and diluted with three volumes of Beltsville Thawing Solution (BTS) and immediately divided into a control group without addition of antioxidants (CTR) and the APS group. Sperm and antioxidant parameters were evaluated in the CTR and APS groups after 1 h of incubation at 37 °C. Finally, we studied the effect of APS (0.5 mg/mL) supplementation on in vitro fertilization (IVF) and embryo development. The addition of different doses of APS to thawed sperm did not induce any significant effects on the sperm viability or motility compared to the sperm without APS treatment. However, the addition of 0.5 mg/mL APS to thawed sperm showed improved mitochondrial activity, higher penetration rate and increased total IVF efficiency compared with those of the control group. Moreover, our results indicate that the supplementation of APS in thawed sperm decreased the concentration of reactive oxygen species (ROS) and improved the activity of superoxide dismutase (SOD) and catalase (CAT). Finally, the addition of APS significantly increased the cleavage rate and blastocyst rate compared to those of the control group. In conclusion, the addition of APS to thawed boar sperm can enhance the antioxidant ability of sperm and improve in vitro fertilization (IVF) parameters and the outcomes of embryonic development. These results imply that APS has practical potential to enhance boar sperm reproductive performance.

Heat stress adversely affects the productivity and immune status of dairy cows. The temperature-humidity index (THI) is commonly used to indicate the degree of heat stress on dairy cattle. We investigated the effects of different THI and Cr supplementation on the antioxidant capacity, the levels of heat shock protein 72 (Hsp72), and cytokine responses of lactating cows. The study used a total of 24 clinically healthy uniparous midlactation Holstein cows, which were randomly divided into 2 groups (n = 12 per group), and was conducted in 3 designated THI periods: low THI period (LTHI; THI = 56.4 ± 2.5), moderate THI period (MTHI; THI = 73.9 ± 1.7), and high THI period (HTHI; THI = 80.3 ± 1.0). The 2 groups of cows were fed corn and corn silage based basal diet supplemented chromium picolinate to provide 3.5 mg of Cr/cow daily (Cr+) or basal diet with no Cr (Cr-). The experiment was a 3 × 2 factorial design. The numbers of leukocytes (P < 0.05) and serum levels of glucose (P < 0.001) were lower; however, the serum levels of blood urea nitrogen (BUN; P < 0.001) and creatinine (P < 0.001) were greater in the MTHI and HTHI than in LTHI. The total antioxidant capacity in the serum was unaltered; an increase in superoxide dismutase activity (P < 0.001) and in serum malondialdehyde concentration (P < 0.001) was observed in the MTHI and HTHI compared with the LTHI. The high THI led to increases in serum concentrations of tumor necrosis factor-α (TNF-α; P < 0.001) and IL-10 (P < 0.05). Cows supplemented with Cr had lower (P = 0.009) serum concentrations of cholesterol but greater (P < 0.001, respectively) serum levels of Hsp72 and IL-10 compared with those without Cr supplementation in the HTHI. Western blot analysis revealed that cows supplemented with Cr had greater (P = 0.038) expression of the inhibitor of nuclear factor kappa B α (IκBα) in peripheral blood mononuclear cells (PBMC) compared with those without Cr supplementation in the HTHI, whereas the expression of Hsp72 in PBMC was unaltered. Data indicate that there is a decrease in glucose and increases in BUN and creatinine in the serum of midlactation cows under hot conditions during the summer and that these cows have a lowered oxidative capacity but an elevated antioxidant capacity. In addition, Cr may play an anti-inflammatory role in lactating cows by promoting the release of Hsp72, increasing the production of IL-10, and inhibiting the degradation of IκBα under hot conditions during the summer.

Ya Jie Wang,1 Qi Li,1 Hong Bin Xiao,1 Yu Jie Li,1 Qing Yang,1 Xiao Xi Kan,1 Ying Chen,1 Xiao Ni Liu,2 Xiao Gang Weng,1 Xi Chen,1 Wei Yan Cai,1 Yan Guo,1 He Fei Huang,1 Xiao Xin Zhu11Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, 2Beijing Institute of Hepatology and Beijing Youan Hospital, Capital Medical University, Beijing, People’s Republic of ChinaAbstract: Multidrug resistance (MDR) is the main obstacle limiting the efficacy of cancer chemotherapy. Looking for novel anti-MDR agents is an important way to conquer cancer drug resistance. We recently established that chamaejasmin B (CHB), a natural biflavone from Stellera chamaejasme L., is the major active component. However, its anti-MDR activity is still unknown. This study investigated the anti-MDR effect of CHB and the underlying mechanisms. First, it was found that CHB inhibited the growth of both sensitive and resistant cell lines in vitro, and the average resistant factor (RF) of CHB was only 1.26. Furthermore, CHB also displayed favorable anti-MDR activity in KB and KBV200 cancer cells xenograft mice. Subsequent study showed that CHB induced G0/G1 cell cycle arrest as well as apoptosis both in KB and in resistant KBV200 cancer cells. Further studies showed that CHB had no influence on the level of Fas/FasL and activation of procaspase 8. However, CHB-induced apoptosis was dependent on the activation of caspase 9 and caspase 3. Moreover, CHB treatment resulted in the elevation of the Bax/Bcl-2 ratio, attenuation of mitochondrial membrane potential (ΔΨm), and release of cytochrome c and apoptosis-inducing factor from mitochondria into cytoplasm both in KB and KBV200 cells. In conclusion, CHB exhibited good anti-MDR activity in vitro and in vivo, and the underlying mechanisms may be related to the activation of mitochondrial-dependant intrinsic apoptosis pathway. These findings provide a new leading compound for MDR therapy and supply a new evidence for the potential of CHB to be employed in clinical trial of MDR therapy in cancers.Keywords: drug resistance, chamaejasmin B, apoptosis, anticancer, Bax/Bcl-2

The aim of this research is to investigate the protection of PM2.5 infected RAW264.7 cell by traditional Chinese medicine (TCM)--Shenlian(SL) extracts and to establish the damage model. We use cell growth, cell damage and oxidative stress related markers, and inflammatory cytokines as observation index to evaluate the protection of PM2.5 infected RAW264.7 by SL extract. The results showed that 50 mg x L(-1) PM2.5 could cause cell particle deposition, inhibit the growth of cells, and significantly increase the cell supernatant of LDH, NO release quantity and intracellular reactive oxygen species (ROS) level during 4 h and 24 h. In the intervention of SL extract 50, 25, 10 mg x L(-1), the particle deposition of RAW264.7 cells, cell supernatant of LDH, NO, IL(-1) beta release, MCP-1 was significantly decreased, the SOD activity increased significantly. It shows that SL extracts of PM2.5 infected RAW264.7 cell damage has obvious protective effect, the effect may be related to the direct protection of cells, reduce oxidative stress and inflammatory injury.

Multi-drug resistance (MDR) is one of the major obstacles to successful chemotherapy for tumors. Traditional Chinese medicine that can reverse MDR has been intensely studied because of its low toxicity, high efficacy and multi-targets. In recent years, more and more traditional Chinese medicine (TCM) has been found to be effective in reversing MDR. In this review, we analyze the current status of traditional Chinese medicine on reversing tumor MDR and describe recent progress on it.

With the increasingly more serious environmental pollution in China in recent years, effective intervention with PM25-induced health risks has become a major scientific issue to be addressed urgently in medical research field in China. NOD-like receptors (NLRs) are a family of cytoplasmic pattern-recognition receptors that have critical roles in innate immunity. On the basis of study progresses in international cardiovascular disease research "Fine particulate matter exposure is a modifiable risk factor for the morbidity and mortality of cardiovascular diseases", and with reference to the current understanding of pulmonary inflammation and oxidative stress in PM2.5-induced acute coronary syndrome, this study intended to investigate whether intracellular pattern recognition NL-RP3 plays a important role in the inital event of PM2.5 induced vessel inflammation as a foreign matter in the process of plaque destabilization and to thoroughly explore the underlying mechanisms responsible for PM2.5-induced acute cardiovascular events. On the other hand, it also studies the feasibility of using traditional Chinese medicine to treat plaque destabilization cause by PM2.5 exposure and discuss it's pathogenesis and intervention strategy based on TCM theory. This paper in order to provide scientific basis for social focal issues in public health proactively and offers the references for relevant research.

This study discusses the effects of Shenlian extracts (SL) on M1 macrophages in atherosclerosis. The MTT assay was used to detect the growth inhibition rates of RAW264.7 cells. RAW264.7 cells were stimulated with murine interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS) to induce M1 macrophages. The different concentrations of SL extracts (high-dose 50 mg x L(-1), moderate-dose 25 mg x L(-1), low-dose 12.5 mg x L(-1)) were added. The CD86 of M1 macrophages in cell membrane was measured by flow cytometry. The mRNA expression of iNOS and TNF-alpha gene was detected by reverse transcription PCR (RT-PCR). And the supernatants were collected, the content of IL-6 and TNF-alpha were detected with ELISA kits. The results of this experiment show that the expression of the cell membrane molecule CD86, iNOS and TNF-alpha gene, the content of IL-6 and TNF-alpha was obviously increased in M1 macrophages by IFN-gamma and LPS. The different doses of SL extract could reduce the expression of the above indicators. The above experimental results demonstrate that IFN-gamma combined LPS can induce RAW264.7 cell to type into M1 macrophages, and SL extracts can inhibit M1 macrophages.

A L9 (3(4)) orthogonal design table to be used to get nine combinations of extraction of three herbs of Wuji pill: Coptis chinensis, Tetradium ruticarpum and Paeonia lactiflora Pall., and nine extraction of single herbs correspondingly, altogether eighteen combinations. Quantification of five representative bioactive ingredients: berberine, palmatine, evodiamine, rutaecarpine, paeoniflorin in rat liver by ultra high liquid chromatography-tandem mass spectrometry after oral administration at 2 h time point of eighteen combinations. The result shows the bioactive ingredients have different concentrations betweem different combinations and the single herb with the same dosage significantly as well as the same dose combinations. C. chinensis with evodiamine concentration of low and high dose T. ruticarpum was positively correlated. T. ruticarpum with berberine concentration of low dose C. chinensis was negatively correlated and of meddle dose C. chinensis was correlated positively. T. ruticarpum with paeoniflorin concentration of middle dose P. lactiflora was correlated positively. P. lactiflora with palmatine concentration of middle dose C. chinensis was negatively correlated and with evodiamine and rutaecarpine concentration of middle dose T. ruticarpum was negatively correlated. These shows the three single herbs interactions resulted in the differences of each ingredients concentration in rat liver. The orthogonal analysis indicates the combination 12: 6: 6 make the maximum concentration in rat liver.

Bovine viral diarrhea virus (BVDV), the causative agent of bovine viral diarrhea/mucosal disease (BVD/MD), is an important pathogen of cattle and other wild animals throughout the world. BVDV infection typically leads to an impaired immune response in cattle. In the present study, we investigated the effect of Forsythoside A (FTA) on BVDV infection of bovine peripheral blood mononuclear cells (PBMCs). We found that Forsythoside A could not only promote proliferation of PBMCs and T cells activation but also inhibit the replication of BVDV as well as apoptosis induced by BVDV. FTA treatment could counteract the BVDV-induced overproduction of IFN-γ to maintain the immune homeostasis in bovine PBMCs. At same time, FTA can enhance the secretion of IL-2. What's more, BVDV promotes the expression of CD28, 4-1BB and TRAF-2, which can be modulated by FTA. Our data suggest that FTA protects PBMCs from BVDV infection possibly via TRAF2-dependent CD28-4-1BB signaling, which may activate PBMCs in response to BVDV infection. Therefore, this aids in the development of an effective adjuvant for vaccines against BVDV and other specific FTA-based therapies for preventing BVDV infection.

The effect of adventitia on the occurrence and development of atherosclerosis (As) is getting more attentions. Fibroblasts, mast cells, dendritic cells, vasa vasorums, vascular-associated lymphoid tissues, and vascular peripheral nerves are related to the occurrence and development of As. This essay summarizes studies on the changes in adventitia in As process and its effect on the occurrence and development of As, as well as the latest progress.

To study the metabolism of berberine and palmatine in prescription compatibility of Wuji Wan in human intestinal flora.The L9 (3(4)) orthogonal design was adopted to compare prescription compatibility of nine groups of Wuji Wan composed of Coptis chinensis, Evodiae and fried Radix paeoniae alba into and single ingredient of C. chinensis. They were cultivated with fresh human excrements under anaerobic conditions for 24 h. A HPLC-UV method was adopted for determining berberine and palmatine in bacteria culture medium, in order to compare the metabolism of the two components in different prescription compatibility.Metabolism of berberine was positively correlated with doses, whereas metabolism of palmatine was negatively correlated with doses in extracts from C. chinensis. Compound compatibility speeded up the metabolism of berberine in low dose, which was positively related to the doses of Evodiae and fried Paeoniae Alba Radix; meanwhile Compound compatibility slowed down the metabolism of berberine in high dose, which was negatively related to the dose of Evodiae. Compound compatibility speeded up the metabolism of palmatine in high dose, which was negatively related to the doses of Evodiae and fried Paeoniae Alba Radix.The metabolism of the compatibility of Wuji Wan speeds up, when Coptis chinensis components metabolite rapidly in intestinal flora; while the metabolism of the compatibility of Wuji Wan slows down, when C. chinensis components metabolite slowly in intestinal flora. Therefore, they show a balanced effect. Additionally, different proportion of C. chinensis, Evodiae and fried Paeoniae Alba Radix cause difference in metabolism speed of berberine and palmatine to some extent.

In this study, we aimed to investigate the therapeutic effects and involved mechanisms of resveratrol on an established non-alcoholic fatty liver disease (NAFLD) murine model. Wild-type and autophagic mediator ULK1 heterozygous knockout mice were induced to have NAFLD by high-fat diet for 8weeks. After that, resveratrol treatment was applied with the high-fat diet feeding for another 4weeks. Typical features of NAFLD, including histological changes, fibrosis, insulin resistance, oxidative status, and inflammation were characterized. After-treatment with resveratrol showed ameliorative effects on all measured features of NAFLD, from histology, insulin resistance, glucose tolerance to oxidative stress and inflammation. resveratrol treatment also reduced the activity of nuclear factor-κB (NF-κB) through the restoration of its inhibitor IκBα. Partial inhibition of ULK1 expression impaired the ameliorative effects of resveratrol on hepatic histology, fibrosis, oxidative status, inflammation, and NF-κB activity. In conclusion, resveratrol improved NAFLD-caused hepatic injury partially through regulating autophagic and IκBα-NF-κB pathways.

Background Astragalus polysaccharide (APS) has been used as an immunomodulator that can enhance immune responses, whereas the immunomodulatory effects of APS on porcine peripheral blood mononuclear cells (PBMCs) exposed to porcine reproductive and respiratory syndrome virus (PRRSV) and classical swine fever virus (CSFV) have not been investigated. Methodology/Principal Findings Porcine PBMCs were cultured in complete RPMI media in the presence of the R98-strain of PRRSV (5×104 TCID50/ml) or C-strain of CSFV (103 TCID50/ml) with or without APS. The expression of mRNA for CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), transforming growth factor-β (TGF-β), interleukin 2 (IL-2) and IL-10 was assayed by TaqMan real-time RT-PCR. The expression of mRNA for CD28 and CTLA-4 increased at 24 h after stimulation of PBMCs with CSFV and the increased production of CTLA-4 was confirmed by western blot analysis, whereas the increases were inhibited by the addition of APS. In addition, APS alone upregulated IL-2 and TGF-β mRNA expression in PBMCs and the addition of APS had the capacity to prevent a further increase in IL-2 mRNA expression in PBMCs during CSFV or PRRSV infection, but had no effect on TGF-β mRNA expression. The production of tumor necrosis factor-alpha (TNF-α) increased at 12 h after stimulation with PRRSV or CSFV, but not with PRRSV plus APS or CSFV plus APS, whereas the addition of APS to PBMCs infected with PRRSV or CSFV promoted IL-10 mRNA expression. Conclusions We suggested that APS had immunomodulatory effects on cells exposed to PRRSV or CSFV. It might be that APS via different mechanisms affects the activities of immune cells during either PRRSV or CSFV infection. This possibility warrants further studies to evaluate whether APS would be an effective adjuvant in vaccines against PRRSV or CSFV.

Pheromonicin-SA (Ph-SA) is a newly developed, engineered multidomain peptide that has a bactericidal effect against Staphylococcus aureus. The objective of this study was to characterize innate immune responses by Staph. aureus-stimulated bovine mammary epithelial cells (BMEC) following treatment with Ph-SA. Primary BMEC from one lactating Holstein cow were isolated and exposed to Staph. aureus for 2 h, and then treated with rifampicin or Ph-SA. Total RNA was isolated from BMEC at 0, 2, 6, 12, and 24 h postinfection, and the mRNA expression of selected genes, including toll-like receptor (TLR)2 and TLR4, IL-1β, IL-6, IL-8, tumor necrosis factor α (TNF-α), and lactoferrin, was quantified by real-time PCR. In the rifampicin group, increases in the expression of mRNA for TNF-α, IL-1β, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection and in the expression of mRNA for TLR2 but not for TLR4 at 12 h postinfection. In the Ph-SA group, increases in the mRNA expression of TLR2, TNF-α, IL-1β, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection, and an increase in TLR4 mRNA expression was observed at 24 h postinfection. At 24 h postinfection, the mRNA expression of TLR4, TNF-α, IL-1β, IL-6, IL-8, and lactoferrin was higher in the Ph-SA group than in the rifampicin group. In conclusion, Ph-SA might promote the expression of mRNA for TLR2, TLR4, the pro-inflammatory cytokines IL-1, IL-6, and TNF-α, the chemotactic factor IL-8, and lactoferrin in Staph. aureus-infected BMEC. Moreover, Ph-SA may be of value as an antibiotic in promoting innate immune responses by Staph. aureus-infected bovine mammary epithelial cells.

To establish fingerprint of Cortex Fraxini and provide reference for quality evaluation of Cortex Fraxini.Chromatographic experiments were performed on Agilent Extend C18 column (4.6 mm x 250 mm, 5 microm), eluted with methanol and water, containing 0.4% acetic acid as the mobile phases in gradient elution. The detection wavelength was 0-60 min, 340 nm; 6-74 min, 254 nm; 74-75 min, 340 nm, and the flow rate was 1.0 mL x min(-1). Forty samples in four varieties of Cortex Fraxini were detected to establish fingerprints, respectively.Every parameter of the method validation complied with related rules and regulations. There were 15 common peaks in the fingerprint of 10 Fraxinus rhynchophylla samples, eleven common peaks in the fingerprint of 10 F. chinensis var. acuminata. samples, and in the fingerprint of 10 F. chinensis samples. Nineteen common peaks in the fingerprint of 10 F. stylosa samples. There were 5 common peaks in the fingerprints of 40 Cortex Fraxini samples. The similarity factors of the 10 samples of every species were all more than 0.96 compared with the control fingerprint. The similarity of the 40 Cortex Fraxini samples was more than 0.90. Four effective constituents and one unknown constituent were found in 40 samples.The fingerprints of F. rhynchophylla bark, F. chinensis bark, F. chinensis var. acuminata bark, F. stylosa bark and Cortex Fraxini were established. The methodological evaluation showed that the results were in accord with the technology requirements of chromatography fingerprint, and it laid a good foundation for quality control of Cortex Fraxini.

Using in vitro everted gut seas to research the intestinal absorption of the extractive Rhizoma Coptidis at the different intestinal section and the different density.Berberine (BER) and palmatine (PAL) which are representative compositions of the extractive Rhizoma Coptidis in everted gut seas are detected by HPLC, and calculated the absorption parameter to describe the character of absorption.The absorption of BER and PAL is linearity in different intestine and different dose, and the square of coefficient correlation exceed 0.9, which consistent with zero order rate process. The K(a) of BER and PAL increases along with the raised dosage of the extractive Rhizoma Coptidis (P0.05), indicated it is the passive absorption. The absorption of BER and PAL in the jejunum is the most quick, the ileum and colon are slower.In the different dosage of extractive Rhizoma Coptidis, the absorption of BER and PAL Conforms to the zero order rate process at the different intestine, and is the passive absorption.

To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5'-untranslated region (5'-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-α (IFN-α), IFN-β, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-α, and IFN-β mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.

Clonidine-displacing substance (CDS) is a potent stimulator of insulin release from pancreatic beta-cells and has been suggested to constitute the endogenous ligand for the islet imidazoline-binding site. Here we have explored the effects of CDS on glucagon release from mouse pancreatic alpha-cells. CDS (5 U/ml) produced a 35% inhibition (P0.05) of glucagon release from intact islets. This effect was dose-dependent and half-maximal inhibition by CDS was observed at 0.03 U/ml. Inhibition of glucagon release was not associated with a change in whole-cell ATP-sensitive K(+)-channel activity in single alpha-cells. However, during intracellular application through the recording pipette, CDS produced a 36% (P0.05) decrease in the rate of exocytosis, measured as changes in cell capacitance. The inhibitory effect of CDS on exocytosis resulted from activation of the protein phosphatase calcineurin and was abolished by cyclosporin A. These data provide further evidence for a role of CDS as an endogenous ligand controlling islet hormone secretion.

Xue-Hui Chen, Zhi-Qiang Li, Hua Peng, Su-Mei Jin, Hui-Qing Fu, Tie-Chui Zhu, Xiao-Gang WengThe First Affiliated Hospital of Xinxiang Medical University, Weihui, People's Republic of ChinaAbstract: One of the most important molecules mediating the proliferation, growth, and metastasis of cancer cells is insulin-like growth factor (IGF), with its receptor IGF-R1. Here, we describe the potential of an IGF-1R monoclonal antibody, HX-1162, on liver cancer apoptosis in vitro and in vivo. We found that HX-1162 could induce the apoptosis of cultured liver cancer cells. Additionally, HX-1162 treatment inhibited the tumor growth after cancer cell grafting and enhanced the cell apoptosis inside the tumor tissue. We conclude that IGF-R1 targeting therapy provides a new avenue toward treating liver cancer.Keywords: IGF, IGF-R1, apoptosis, hepatocellular carcinoma