OBJECTIVETo demonstrate the vasodilatation activity of the coumarin-containing Angelica dahurica var. formosana and to further analyze active components in the herb extracts.

METHODS(1) The vasodilatation effects induced by different extracts (cyclohexane, ethyl acetate, acetone, methanol, 95 % ethanol and water) of Angelica dahurica var. formosana on mouse thoracic aorta pre-contracted with phenylephrine were investigated. (2) The amount of imperatorin and isoimperatorin in each extract was measured by high-performance liquid chromatography. (3) The vasodilatation effects of imperatorin and isoimperatorin on mouse thoracic aorta were compared using the same in vitro method. (4) The vasodilatation mechanism of imperatorin in the mouse thoracic aorta pre-contracted with phenylephrine was studied using the methods of denuded endothelium, NG-nitro-L-arginine methylester (L-NAME, a nitric oxide synthase inhibitor), and propranolol.

RESULTS(1) The cyclohexane and ethyl acetate extracts of Angelica dahurica var. formosana decreased the maximal response of phenylephrine-induced mouse thoracic aorta contraction dose-dependently, with 50% inhibiting concentration (IC(50)) values of 35.3+/-12.4 mg/L and 40.5+/-12.0 mg/L, respectively. The vasodilatation effect of imperatorin and isoimperatorin was dose-dependent. (2) The cyclohexane extract, showing the strongest vasodilatation effect, possessed the highest contents of imperatorin (4.09%) and isoimperatorin (0.27%, w/w). There was a correlation between the vasodilatation activity and the contents of imperatorin and isoimperatorin in the extracts. (3) The vasodilatation effect of imperatorin was about 4-fold stronger than that of isoimperatorin. (4) The vasodilatation effect of imperatorin was signifificantly attenuated to 24.88%+/-4.06% in the denuded endothelium group compared with the intact endothelium group. And 1 mmol/L L-NAME reduced the imperatorin-induced vasorelaxation by 32.18 %+/-11.29 %.

CONCLUSIONSThe principal effective component of Angelica dahurica var. Formosana was found to be imperatorin. Imperatorin-induced vasodilatation is at least partially regulated by nitric oxide, and has no correlation to beta-receptor.

Angelica ; chemistry ; Animals ; Chromatography, High Pressure Liquid ; Endothelium, Vascular ; physiology ; Furocoumarins ; analysis ; pharmacology ; Male ; Mice ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; physiology ; Phenylephrine ; pharmacology ; Plant Extracts ; pharmacology ; Propranolol ; pharmacology ; Vasodilation ; drug effects

Objective To observe the improvement effect and mechanism of salvianolic acid B on renal fibrosis mice.Methods Thirty-six BALB/c mice were randomly divided into sham operation group,model group,salvianolic acid B low-,medium-and high-dose groups and VX765(caspase-1 inhibitor)group,with 6 mice in each group.Salvianolic acid B low-,medium-and high-dose groups were intragastrically administered with corresponding doses of 50,100,200 mg-kg-1-d-1 salvianolic acid B normal saline suspension,respectively,VX765 group was intragastrically administered with 50 mg·kg-1·d-1 VX765 normal saline suspension,and the sham operation group and the model group were intragastrically administered with the same volume of normal saline,once a day.After seven days,except for the sham operation group,the mice in other groups were treated with unilateral renal ischemia-reperfusion injury combined with contralateral nephrectomy to establish an acute kidney injury-induced renal fibrosis model.After modeling,each group continued to be administered with the corresponding volume of drugs for 21 days.After the administration,the pathological changes of renal tissue were observed by hematoxylin-eosin(HE)staining and Masson staining.The serum creatinine(SCr)was detected by sarcosine oxidase method.The urea nitrogen(BUN)was detected by urease method.The level of serum neutrophil gelatinase-associated lipocalin(NGAL)was detected by enzyme-linked immunosorbent assay(ELISA).The expression of fibrosis-related proteins fibrosis-related proteins α-smooth muscle actin(α-SMA),vimentin and transforming growth factor-β(TGF-β)and pyroptosis-related proteins cleaved cysteine aspartate proteolytic enzyme 1(cl-caspase-1),pro-caspase-1(pro-caspase-1),NOD-like receptor hot protein domain-related protein 3(NLRP3),cleaved interleukin-1β(cl-IL-1β),interleukin-1β(IL-1β),GSDMD-N,GSDMD in renal tissue was detected by Western Blot.Results Compared with the sham operation group,the serum levels of SCr,BUN and NGAL in the model group were increased(P＜0.05 or P＜0.01).HE and Masson staining showed inflammatory cell infiltration and a large amount of collagen deposition in the renal interstitium,and the expression levels of fibrosis-related proteins α-SMA,vimentin and TGF-β were increased(P＜0.01).The expression levels of pyroptosis-related proteins cl-caspase-1/pro-caspase-1,NLRP3,cl-IL-1β/IL-1β,GSDMD-N/GSDMD were also increased(P＜0.05 or P＜0.01).Compared with the model group,the levels of serum SCr,BUN and NGAL in the high-dose salvianolic acid B group were significantly decreased(P＜0.05 or P＜0.01),the infiltration of renal interstitial inflammatory cells and collagen deposition were reduced,and the expression levels of fibrosis proteins α-SMA,vimentin,TGF-β and pyroptosis-related proteins cl-caspase-1/pro-caspase-1,NLRP3,cl-IL-1β/IL-1β,GSDMD-N/GSDMD were decreased(P＜0.05 or P＜0.01).There was no significant difference in the above indexes between the high-dose salvianolic acid B group and the VX765 group(P＞0.05).Conclusion Salvianolic acid B may alleviate renal fibrosis in mice by inhibiting pyroptosis mediated by NLRP3/caspase-1/GSDMD pathway.

To express and secrete native HBscFv (anti-HBsAg single-chain Fv) in P. pastoris, HBscFv was amplified from plasmid pGEM-HBscFv, and then sub-cloned into expression vector pPICZalphaA. The resulting plasmid pPIC-HBscFv was linearized and transformed into P. pastoris GS115. The recombinant Pichia strains, identified by direct PCR and Zeocin-resistant screening of Pichia transformants, were cultured and induced with methanol. It was found that recombinant HBscFv, lead by alpha-factor, could be secreted into the culture supernatant to a level of 80mg/L. The bioactivity of Pichia produced HBscFv was confirmed by indirect ELISA, which also suggested that the bioactivity of HBscFv in the culture supernatant reached its peak in 72h and decreased in the late-stage of the induction. PAS staining suggests that HBscFv produced by yeast is poorly glycosylated or none-glycosylated protein.
Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Hepatitis B Surface Antigens ; immunology ; Humans ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Single-Chain Antibodies ; genetics ; immunology ; metabolism

Using beta-2 adrenergic receptor, 5-hydroxytryptamine and angiotensin II type 1 receptor as control, we here established a method for rapid prediction of the initial position amino acids of N-terminal, C-terminal, intracellular loops, extracellular loops and transmembrane (TM) regions in G protein-coupled receptors (GPCRs), and successfully predicted the structure of Mas-related G protein-coupled receptors X3 (MRGPRX3). To achieve this purpose, nanoluciferase (Nluc) was inserted into the different sites of these GPCRs′ sequence by sequence and ligation-independent cloning (SLIC) method, and the luminescence value were measured to distinguish the different parts of GPCRs. The results showed that luminescence values of NLuc luciferase at TM region were less than 100 000, and the values were higher than 1 000 000 at N terminal, C terminal, or extracellular loops and intracellular loops, and the values were between 100 000 and 500 000 at junction. The predicted MRGPRX3 structure was analyzed in detail and was compared with AlphaFold predicted structure. In conclusion, this method could provide useful information of GPCR structure model for the ligand virtual screening, and could provide certain experimental basis for structural pharmacology.

OBJECTIVETo investigate the underlying mechanisms of cyclovirobuxinum D (Cvb-D) on alleviating cardiac hypertrophy in rats.

METHODSSprague-Dawley rats were randomly divided into 5 groups: control group; levothyroxine-induced cardiac hypertrophy group (model); levothyroxine-induced cardiac hypertrophy + Cvb-D group (Cvb-D); levothyroxine-induced cardiac hypertrophy + captopril group (captopril); levothyroxine-induced cardiac hypertrophy + SB203580 group (SB203580), n=10 for each group. Rats were daily administered the respective drugs continuously for14 days by gastric gavage. A rat model of cardiac hypertrophy was established by intraperitoneal injection of levothyroxine to investigate whether Cvb-D protects against cardiac hypertrophy by inhibiting the p38 mitogen-activated protein kinase (MAPK) signaling pathway and preventing apoptosis of cardiac cells.

RESULTSTreatment with Cvb-D significantly deceased left ventricle hypertrophy, improved the histopathology, hemodynamic conditions, and cardiac function in rats with cardiac hypertrophy. Compared with the normal control group, in rats with cardiac hypertrophy, expression of bax in the heart and phospho-p38 MAPK protein levels were significantly up-regulated (P<0.01 or 0.05), whereas the bcl-2 protein level was down-regulated (P<0.01). In contrast, Cvb-D treatment reversed the changes in bax and phospho-p38 MAPK protein levels but increased the bcl-2 protein level (P<0.01 or 0.05), and these effects were similar to those of captopril and SB203580 (a specific p38MAPK inhibitor) treatment. Furthermore, both Cvb-D, captopril and SB203580 reduced mRNA expression of p38α, p38β, c-fos, and c-jun mRNA, and Cvb-D had a stronger effect (P<0.01).

CONCLUSIONThese results demonstrate that Cvb-D protects against cardiac hypertrophy, which is possibly mediated by prevention of cardiac cell apoptosis and inhibition of the p38MAPK signaling pathway.