Objective To lay the foundation of new botanical pesticides by screening the insecticidal efficacy of 10 commonly used TCM pesticides.Methods Slide immersion method, trace intravenous drip method and insect body dipping method were respectively used to measure contact action of water extract and ethanol extract of the 10 commonly used TCM pesticides toTeranychus cinnbarinus,Brevicoryne brassicaeandMythimna separate, with herbal extracts to three kinds of insect pests of corrected mortality as observation indexes,Results Alcohol extracts for three kinds of insect pests were found to have contact action, and water extracts had no obvious effect. The Stemonae Radix alcohol extract had obvious contact action on three kinds of insect pests, and corrected mortality rates were 78.7%, 85.9%, and 96.7%, respectively; ethanol extracts of Angled Bittersweet, Sophorae Flavescentis Radix, Xanthii Fructus and Kansui Radix showed good contact action to Teranychus cinnbarinus,. The volume ratio of compatibility among Angled Bittersweet, Sophorae Flavescentis Radix, Xanthii Fructus and Kansui Radix was 2:1:1.5:1.5:1.5:1.5. The insecticidal activity to Teranychus cinnbarinus, was stronger than extracts of single herbs, and corrected mortality was 96.5%. Alcohol extracts of Stellera, hellebore, white mustard seed, and Xanthii Fructus showed good contact action toBrevicoryne brassicae. When the volume ratio of compatibility among Xanthii Fructus, Stemonae Radix, Stellera, and hellebore was 1:1.5:2:2, the activity toBrevicoryne brassicae increased. and corrected mortality was 93.1%.Conclusion The insecticidal activity will be enhanced after TCM extracts are under compatibility.

Abstract: In order to investigate immune protection against swine-origin influenza virus (S-OIV) A H1N1, the helper-dependent adenovirus vector (HDAd) system was exploited to construct recombinant HDAd encoding hemagglutinin (HA). The HA gene was synthesized and cloned to the HDAd backbone. Then, the HDAd/HA DNA molecules were transfected into 293Cre4 cells with calcium phosphate. The cells were infected by helper virus 16 hours after the transfection. The 293Cre4 cells were coinfected with HDAd/HA and the helper virus for large-scale preparation of HDAd/HA. The HDAd/HA was obtained and purified twice with CsCI density ultracentrifugation and observed morphologically under transmission electron microscope, and the expression of HA protein was analyzed with RTPCR. Recombinant HDAd/HA expressing HA protein was successfully constructed which could pave the way for in vivo investigation on immunogenicity and efficacy against S-OIV A H1N1 infection.
Adenoviridae ; Cell Line ; Cloning, Molecular ; Genetic Vectors ; Helper Viruses ; Hemagglutinin Glycoproteins, Influenza Virus ; biosynthesis ; Humans ; Influenza A Virus, H1N1 Subtype

Objective To compare analgesic and anti-inflammatory effects of Gentianopsis Paludosa and Sophora Alopecuroide of different kinds of compatibility on the mice.Methods Mice were randomly divided into normal control group, positive medicine group, and different kinds of compatibility groups, 10 mice in each group. Mice received gavage for successive 7 days, once a day. Glacial acetic acid twist body method and hot board to pain method were used to observe and compare analgesic effects of different kinds of compatibility. Xylene to ear swelling method and agar to granulation swollen method were used to observe and compare inflammatory effects of different kinds of compatibility.Results Compared with normal group, compound Gentianopsis Paludosa had obvious analgesic and anti-inflammatory effects (P<0.01). When the ratio of Gentianopsis Paludosa and Sophora Alopecuroide was 20:0.6 and 20:0.9, the analgesic and anti-inflammatory effects were the best.Conclusion Gentianopsis Paludosa and Sophora Alopecuroide have good analgesic and anti-inflammatory effects, among which, when the ratio of Gentianopsis Paludosa and Sophora Alopecuroide is 20:0.6, the best effects show.

Objective We aimed to investigate the effects of Biejiajian Pills on MHCC-97H hepatoma cells and whether Biejiajian Pills regulate the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway through miR-885-5p.Methods SPF SD rats (n = 10) were randomly divided into the blank group and the Biejiajian Pills (1.1 g/kg) group to prepare blank and Biejiajian Pills-containing serum. MHCC-97H cells in the logarithmic growth phase were divided into the model group, blank serum groups with different concentrations (5％, 10％, 15％, and 20％), the serum containing Biejiajian Pills group, and the blank group without cells. Cell proliferation was detected by the CCK-8 assay, and the optimal intervention time and concentration of drug-containing serum were screened. MHCC-97H cells were divided into the blank control group (no intervention), the Biejiajian Pills-containing serum group (20％ Biejiajian Pills-containing serum), the miR-885-5p mimics group (transfected with miR-885-5p mimics), the miR-NC group (transfected with miR-885-5p NC), and the Biejiajian Pills-containing serum + miR-885-5p mimics group (treated with 20％ Biejiajian Pills-containing serum and transfected with miR-885-5p mimics). Cells in each group were cultured for 72 hours. A dual luciferase reporter assay was conducted to verify the targeting relationship between miR-885-5p and EGFR. Cell proliferation was detected by the CCK-8 assay, cell migration and invasion abilities were detected by the cell scratch assay and the Transwell invasion assay. Annexin V-APC/PI double staining was performed to detect the apoptosis level, and real-time fluorescence quantitative PCR (RT-qPCR) analysis was conducted to determine the mRNA expression levels of miR-885-5p, EGFR, MEK, and ERK1/2. The expression levels of EGFR, p-EGFR, MEK, p-MEK, ERK1/2, p-ERK1/2, matrix metalloproteinase 1 (MMP1), and CyclinD1 were determined by Western blotting analysis. The subcutaneous tumor model of MHCC-97H hepatoma cells in nude mice was established by subcutaneous injection to observe the inhibitory effect of Biejiajian Pills of different doses(0.55,1.1,2.2 g/kg).Results The optimal concentration and intervention time of Biejiajian Pills-containing serum were 20％ and 72 hours, respectively. Meanwhile, the dual luciferase reporter assay showed that miR-885-5p could directly target EGFR. No statistical significances between the blank control group and the miR-NC group were observed (P>0.05). Compared with the blank control group, the proliferation rates of MHCC-97H hepatoma cells in the Biejiajian Pills-containing serum group, the miR-885-5p mimics group, and the Biejiajian Pills-containing serum + miR-885-5p mimics group were significantly decreased (P<0.01), and their migration and invasion abilities were significantly decreased (P<0.05, P<0.01). At the same time, the protein expression levels of CyclinD1 and MMP1, which are closely related to cell proliferation and invasion, were significantly downregulated (P<0.05, P<0.01). The proportions of late apoptotic cells and the proportion of total apoptotic cells were significantly increased (P<0.01). In the Biejiajian Pills-containing serum group, the miR-885-5p mimics group, and the Biejiajian Pills-containing serum + miR-885-5p mimics group, miR-885-5p mRNA was significantly upregulated (P<0.01) and EGFR, MEK, and ERK1/2 were significantly downregulated at the mRNA level (P<0.05, P<0.01). EGFR, MEK, and ERK1/2 phosphorylation was inhibited (P<0.01), and the Biejiajian Pills-containing serum + miR-885-5p mimics group showed the best effect (P<0.05, P<0.01). The subcutaneous liver tumor model in nude mice verified that Biejiajian Pills can inhibit tumor growth in a dose-dependent manner. Conclusion Biejiajian Pills can promote apoptosis of MHCC-97H hepatoma cells and inhibit their proliferation, invasion, and migration. The mechanism may be related to the targeted regulation of the EGFR/MAPK/ERK signaling pathway by miR-885-5p.

The growth of fibroblasts on the acellular dermal matrix (ADM) was studied. The fibroblasts isolated from the skin of an adult New Zealand Rabbit were cultured in vitro and identified subsequently. After the cells were inoculated on the ADM as seeds, the adhesion rate and the growth ability were examined, and cellular morphology was assayed with DAPI fluorescent staining and Scanning electron microscope (SEM). The possibilities of applying ADM as cells carrier or deliverer in the field of transplantation were evaluated. The result revealed that pure fibroblasts were isolated through the specific method. Skin fibroblasts could adhere to ADM easily, and the adhesion rate was 96.78%, displaying no significant difference (P > 0.05) when compared with that rate of the control holes. The cells on the scaffolds and those on the control holes showed similar growth tendencies, but the activity of the former was lower (P < 0.01). The integral nucleus with blue fluorescence could be observed on the ADM under fluorescence microscope. The number of fibroblasts scaled up with the cultured time, The results of SEM showed that the state of cell was good and the fibroblasts were fused into a layer after being cultured for 5-10d. So rabbit fibroblasts can attach, survive, grow and proliferate on the ADM in a healthy way. It is entirely possible to use ADM as an appropriate scaffold material for the culture of fibroblasts and as a material for transplantations.
Animals ; Biocompatible Materials ; Cell Adhesion ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dermis ; cytology ; Fibroblasts ; cytology ; Rabbits ; Skin ; cytology ; Skin, Artificial ; Tissue Engineering ; methods ; Tissue Scaffolds

Objective:To evaluate the reliability and validity of the Reward Probability Index-Chinese version (C-RPI) among individuals with opioid dependence.Methods:A total of 160 individuals with opioid dependence were recruited from the methadone maintenance treatment(MMT) programs in Wuhan.The C-RPI was used in the investigation, and items/exploratory factors and confirmatory factors were adopted to analyze with IBM SPSS 22.0 and AMOS 24.0, respectively.Results:The correlation between each item and the total score of the scale was 0.325-0.657 (all P<0.01) and the item discrimination of the scale was good.Exploratory factor analysis extracted two factors, including reward probability and environmental suppressors, and a load of each item on its factor ranged from 0.519 to 0.744.The fitting indexes of confirmatory factor analysis were as follows: χ 2/ df=1.623, RMR=0.030, RMSEA=0.063, IFI=0.902, TLI=0.885, CFI =0.900.The internal consistency coefficient (Cronbach's α) of the C-RPI was 0.800; and there was no ceiling/floor effect on each dimension and total scale. Conclusion:The classification of dimensions for the C-RPI is reasonable, and the scale has good reliability and validity to measure the ability to obtain environmental rewards for individuals with opioid dependence.

ObjectiveTo observe the therapeutic effect of Radix Paeoniae Rubra-Radix Aconiti Lateralis on acute-on-chronic liver failure （ACLF） rats and its effect on M1/M2 macrophage polarization. MethodMale SD rats were randomly divided into normal group， model group， positive group （lactulose， 1.8 g·kg^-1） and traditional Chinese medicine （TCM） group （Radix Paeoniae Rubra-Radix Aconiti Lateralis， 5.85 g·kg^-1）， six in each group. The ACLF rat model was established by subcutaneous and tail vein injection of bovine serum albumin combined with intraperitoneal injection of D-galactosamine+lipopolysaccharide. Then the modeled rats were intervened with corresponding drugs for one week. The normal group and model group were given the same dose of distilled water. The histopathological changes of liver tissue were observed by hematoxylin-eosin （HE） staining. The mRNA and protein expression levels of CD86， inducible nitric oxide synthase （iNOS）， CD206 and arginase 1 （Arg1） were detected by real-time polymerase chain reaction （Real-time PCR）， Western blot and immunohistochemistry. ResultCompared with the conditions in the normal group， pseudolobule formation in liver tissue and morphological changes and necrosis of hepatocytes were observed in ACLF rats， accompanied by a large number of inflammatory cell infiltration. Moreover， the mRNA and protein expression levels of CD86， iNOS were up-regulated（P<0.01）. Compared with the model group， the treatment groups had improved necrosis and inflammatory infiltration of hepatocytes， down-regulated mRNA and protein expression of CD86 and iNOS （P<0.01） and up-regulated mRNA and protein expression of CD206 and Arg1 （P<0.05， P<0.01）， with the up regulation in the TCM group better than that in the positive group. ConclusionACLF rats had unbalanced M1/M2 macrophage polarization， and the imbalance shifted towards M1. Radix Paeoniae Rubra-Radix Aconiti Lateralis inhibited the activation of M1 macrophages and reduced the inflammatory response of liver failure by promoting the polarization of liver macrophages towards M2.

ObjectiveTo study the regulatory effects of Yinchenhao Tang combined with Yinchen Zhufu Tang on the expression of regulatory T （Treg）/helper T 17 （Th17） cells cultured in vitro from the patients with hepatitis B virus-related acute-on-chronic liver failure （HBV-ACLF）. MethodsFresh peripheral blood was collected from the patients with HBV-ACLF for the separation of peripheral blood mononuclear cells （PBMCs）. Immunomagnetic beads were used to isolate primary Treg and naive CD4⁺ T cells. After in vitro expansion， naive CD4⁺ T cells were induced to differentiate into Th17 cells. Rats were treated with the clearing method （Yinchenhao Tang）， warming method （Yinchen Zhufu Tang）， and combination of clearing method with warming method （Yinchenhao Tang combined with Yinchen Zhufu Tang， also known as Wenyang Jiedu Huayu Prescription）， respectively， and then the medicated plasma samples were collected. Meanwhile， blank plasma was collected from the rats treated with normal saline. Cells were classified into blank， clearing method （5.04 g·kg^-1）， warming method （6.21 g·kg^-1）， and combination of clearing method with warming method （17.1 g·kg^-1） groups and treated with corresponding plasma. The frequency of Treg/Th17 cells was detected by flow cytometry. The level of transforming growth factor-β （TGF-β） was measured by the enzyme-linked immunosorbent assay. The cytometric bead array （CBA） was employed to measure the levels of interleukin-10 （IL-10）， interleukin-17A （IL-17A）， tumor necrosis factor-α （TNF-α）， and interleukin-23 （IL-23）. The mRNA and protein levels of Forkhead box P3 （FoxP3） and retinoic acid-related orphan receptor-gamma t （ROR-γt） were determined by Real-time PCR and Western blot， respectively. ResultsCompared with the blank group， the combination of clearing method with warming method group showed decreased frequency of Treg and Th17 cells， lowered levels of Treg cytokines （TGF-β and IL-10） and Th17 cytokines （TNF-α， IL-17A， and IL-23）， and down-regulated mRNA and protein levels of FoxP3 and ROR-γt （P<0.01）. Compared with the clearing method group， the combination of clearing method with warming method group showed decreased Treg cell frequency and down-regulated mRNA and protein levels of FoxP3. Meanwhile， the combination group showed decreased Th17 cell frequency， lowered levels of TGF-β， IL-10， TNF-α， IL-17A， and IL-23， and down-regulated mRNA and protein levels of ROR-γt （P<0.05， P<0.01）. Compared with the warming method group， the combination of clearing method with warming method group showed decreased frequency of Treg cells and down-regulated mRNA and protein levels of FoxP3. Meanwhile， the combination group showed decreased Th17 cell frequency， declined levels of TGF-β， IL-10， TNF-α， IL-17A， and IL-23， and down-regulated mRNA and protein levels of ROR-γt （P<0.05）. ConclusionThe combination of clearing method with warming method can down-regulate the expression of specific cytokines of Treg and Th17 cells， inhibit the over activation of Treg and Th17 cells， and reduce the secretion of cytokines such as TGF-β， IL-10， TNF-α， IL-17A， and IL-23， thereby alleviating inflammation and improving the prognosis of the patients with liver failure.

ObjectiveTo investigate the differential expression of intestinal flora in patients with different traditional Chinese medicine （TCM） syndrome types （Yang Huang syndrome， Yin-Yang Huang syndrome， and Yin Huang syndrome） of hepatitis B virus-related acute-on-chronic liver failure （HBV-ACLF） and clarify the biological basis of jaundice and Yin Huang syndrome in liver failure. MethodsA total of 20 cases of HBV-ACLF patients were included in the Yang Huang group， 20 cases in the Yin-Yang Huang group， 16 cases in the Yin Huang group， and 20 healthy adult volunteers. 16S rRNA gene sequencing was used to detect the diversity， species distribution， and differences of the subjects' intestinal flora， and bioinformatics analysis was conducted. ResultsCompared with those in the healthy control group， the species richness and diversity of intestinal flora in the HBV-ACLF Yang Huang group， Yin-Yang Huang group， and Yin Huang group were significantly reduced， and there were significant differences in the composition of intestinal flora compared with healthy volunteers. However， there were no significant differences in the species richness， diversity， and composition of intestinal flora among the three groups. LEfSe analysis showed that compared with the healthy control group， the HBV-ACLF Yang Huang group showed significant enrichment of Staphylococcus aureus（P<0.01）. Yin-Yang Huang group showed significant enrichment of s_Ileibacterium valens（P<0.05，P<0.01）， and the Yin Huang group showed significant enrichment of Enterococcus faecium and Streptococcus sali varius（P<0.05）. These strains may be biomarkers between the three groups of patients and the healthy control group. Compared with that in the Yin-Yang Huang group， Tyzzerella_nexilis was significantly enriched in the Yang-Huang group， and Streptococcus lactiae was significantly enriched in the Yin-Yang Huang group. Compared with that in the Yang-Huang group and the Yin-Yang Huang group， Enterococcus faecalis was significantly enriched in the Yin Huang group. The above strains may be biomarkers among the three groups of patients， and Enterococcus faecium may be a biomarker for the transition from the Yang Huang group to the Yin Huang group. ConclusionsThere are significant differences in the intestinal flora between patients with HBV-ACLF Yang Huang syndrome， Yin-Yang Huang syndrome， and Yin Huang syndrome. Enterococcus faecium is significantly enriched in the Yin Huang syndrome group， suggesting that dysbiosis of the intestinal flora may be the biological basis for jaundice and Yin Huang syndrome in liver failure.

ObjectiveTo study the expression differences of regulatory T cells （Treg） and T helper 17 cells （Th17） in the peripheral blood of patients with hepatitis B virus-related acute-on-chronic liver failure （HBV-ACLF） in five types of traditional Chinese medicine （TCM） syndrome. MethodsA total of 144 patients with HBV-ACLF were included and divided into five types of TCM syndrome， including 34 cases of heat-toxin amassment syndrome， 44 cases of dampness-heat amassment syndrome， 27 cases of Qi-deficiency and stasis jaundice syndrome， 21 cases of spleen-kidney Yang deficiency syndrome， and 18 cases of liver-kidney Yin deficiency syndrome. Meanwhile， 30 healthy volunteers were included as controls. The frequency of Treg and Th17 cells in the peripheral blood of subjects in each group was detected by flow cytometry， and the Treg/Th17 ratio was calculated. Cytometric bead array （CBA） was used to detect the levels of cytokines interleukin （IL）-10， transforming growth factor-β （TGF-β）， tumor necrosis factor-α （TNF-α）， IL-17A， and IL-23. Real-time fluorescence quantitative PCR （Real-time PCR） detected the mRNA expression of forkhead box P3 （FoxP3） and retinoic acid-related orphan receptor gamma t （ROR-γt）. Results（1） Compared with that in the healthy control group， the frequency of Treg and Th17 cells in patients with various TCM syndrome types of HBV-ACLF increased （P<0.05）. Compared with that in the heat-toxin amassment syndrome group， the frequency of Treg and Th17 cells decreased in the dampness-heat amassment syndrome group （P<0.05）， while the frequency of Treg and Th17 cells increased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the dampness-heat amassment syndrome group， the frequency of Treg and Th17 cells increased in the dampness-heat amassment syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the Qi-deficiency and stasis jaundice syndrome group， the frequency of Treg and Th17 cells increased in the spleen-kidney Yang deficiency syndrome group （P<0.05）， while the frequency of Treg and Th17 cells decreased in the liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with the spleen-kidney Yang deficiency syndrome group， the frequency of Treg and Th17 cells decreased in the liver-kidney Yin deficiency syndrome group （P<0.05）. （2） Compared with that in the healthy control group， the Treg/Th17 cell ratio in patients with various TCM syndromes of HBV-ACLF decreased （P<0.05）. Compared with that in the heat-toxin amassment syndrome group， the Treg/Th17 cell ratio increased in the dampness-heat amassment syndrome group （P<0.05）， while it decreased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the dampness-heat amassment syndrome group， the Treg/Th17 cell ratio decreased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the Qi-deficiency and stasis jaundice syndrome group， the Treg/Th17 cell ratio increased in the spleen-kidney Yang deficiency syndrome group and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with the spleen-kidney Yang deficiency syndrome group， the Treg/Th17 cell ratio in the liver-kidney Yin deficiency syndrome group increased （P<0.05）. （3） Compared with those in the healthy control group， the levels of Treg-related cytokines IL-10 and TGF-β， as well as Th17-related cytokines TNF-α， IL-17A， and IL-23， were elevated in patients with various TCM syndrome types of HBV-ACLF （P<0.05）. There was no significant difference in TNF-α levels among different TCM syndrome types. Compared with those in the heat-toxin amassment syndrome group， the levels of IL-10， TNF-β， IL-17A， and IL-23 in the dampness-heat amassment syndrome group， Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome groups increased （P<0.05）. Compared with those in the dampness-heat amassment syndrome group， the levels of IL-10， TGF-β， IL-17A， and IL-23 increased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with those in the Qi-deficiency and stasis jaundice syndrome group， the levels of IL-10， TGF-β， IL-17A， and IL-23 in the spleen-kidney Yang deficiency syndrome group increased （P<0.05）， while those in the liver-kidney Yin deficiency syndrome group decreased （P<0.05）. Compared with those in the spleen-kidney Yang deficiency syndrome， the levels of IL-10， TGF-β， IL-17A， and IL-23 in the liver-kidney Yin deficiency syndrome group decreased （P<0.05）. （4） Compared with that in the healthy control group， the mRNA of Treg/Th17 cell specific transcription factors FoxP3 and ROR-γt were elevated in patients with various TCM syndrome types of HBV-ACLF （P<0.05）. Compared with that in the heat-toxin amassment syndrome group， the mRNA of FoxP3 and ROR-γt increased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the dampness-heat amassment syndrome group， the mRNA of FoxP3 and ROR-γt increased in the Qi-deficiency and stasis jaundice syndrome group， spleen-kidney Yang deficiency syndrome group， and liver-kidney Yin deficiency syndrome （P<0.05）. Compared with that in the Qi-deficiency and stasis jaundice syndrome group， the mRNA of FoxP3 and ROR-γt in the spleen-kidney Yang deficiency syndrome group increased （P<0.05）， and it decreased in the liver-kidney Yin deficiency syndrome group （P<0.05）. Compared with that in the spleen-kidney Yang deficiency syndrome group， the mRNA of FoxP3 and ROR-γt decreased in the liver-kidney Yin deficiency syndrome group （P<0.05）. ConclusionThe frequency and ratio of Treg/Th17 cells， as well as the expression of related cytokines and specific receptors in peripheral blood of patients with HBV-ACLF in five types of TCM syndromes are different， which has certain reference value for TCM syndrome differentiation and treatment of patients with HBV-ACLF.