ObjectiveTo investigate the effect of astragaloside Ⅳ（AS-Ⅳ） on the proliferation of vascular smooth muscle cells（VSMCs） induced by angiotensin Ⅱ（Ang Ⅱ） based on solute carrier family 7 member 11/glutathione peroxidase 4（SLC7A11/GPX4） pathway. MethodsPrimary rat thoracic aortic VSMCs were cultured by tissue explant method， and the cell types were identified by immunofluorescence. Cell counting kit-8（CCK-8） was used to determine the optimal concentration and time of AS-Ⅳ after Ang Ⅱ stimulation. The experiment was divided into blank group， model group， AS-Ⅳ group（40 μmol·L^-1）， Erastin group（0.5 μmol·L^-1）， Erastin+AS-Ⅳ group（0.5 μmol·L^-1+40 μmol·L^-1）. The blank group was cultured in normal medium， the model group was cultured in medium containing Ang Ⅱ（0.1 μmol·L^-1）， and each administration group was cultured in medium containing Ang Ⅱ（0.1 μmol·L^-1） and the corresponding doses of drug. CCK-8 and plate clone formation assay were used to detect the proliferation of cells in each group， Prussian blue staining was used to detect cell iron deposition， the content of reactive oxygen species（ROS） in cells was detected by fluorescence probe method， the content of malondialdehyde（MDA） was detected by thiobarbituric acid（TBA） method， and the protein levels of SLC7A11 and GPX4 in each group were detected by Western blot. ResultsPrimary rat thoracic aortic VSMCs were successfully cultured by tissue explant method， and immunofluorescence detection showed that positive expression of α-smooth muscle actin（α-SMA） and negative expression of vimentin in the cells， identifying them as VSMCs. The optimal concentration and time of AS-Ⅳ determined by CCK-8 were 40 μmol·L^-1 and 24 h， respectively. Pharmacodynamic studies showed that compared with the blank group， the cell proliferation in the model group increased， the iron deposition in the cells increased， the contents of ROS and MDA increased， and the expression levels of SLC7A11 and GPX4 proteins decreased（P<0.05， P<0.01）. Compared with the model group， the cell proliferation of the AS-Ⅳ group was inhibited， the iron deposition in the cells was decreased， the contents of ROS and MDA were decreased， and the expression levels of SLC7A11 and GPX4 proteins were increased（P<0.05， P<0.01）. While in the Erastin group， the cell proliferation was increased， the iron deposition was increased， ROS and MDA contents were increased， and the expression levels of SLC7A11 and GPX4 proteins were decreased（P<0.05， P<0.01）. Compared with the AS-Ⅳ group， Erastin+AS-Ⅳ group showed increased cell proliferation， increased iron deposition in cells， increased ROS and MDA contents， and decreased expression of SLC7A11 and GPX4 proteins（P<0.05）. Compared with the Erastin group， the cell proliferation in Erastin+AS-Ⅳ group was inhibited， the iron deposition was decreased， the contents of ROS and MDA were decreased， and the expression levels of SLC7A11 and GPX4 proteins were increased（P<0.05， P<0.01）. ConclusionAS-Ⅳ can inhibit ferroptosis by regulating the SLC7A11/GPX4 pathway， so as to weaken the proliferation of VSMCs， thus playing a role in the treatment of atherosclerosis.

Humans ; Gastrointestinal Microbiome ; Diabetes Mellitus, Type 2/complications* ; Cholelithiasis

To study the molecular mechanism of salt stress response of peanut small GTP binding protein gene AhRabG3f, a 1 914 bp promoter fragment upstream of the start codon of AhRabG3f gene (3f-P) from peanut was cloned. Subsequently, five truncated fragments (3f-P1-3f-P5) with lengths of 1 729, 1 379, 666, 510 and 179 bp were obtained through deletion at the 5' end, respectively. Plant expression vectors where these six promoter fragments were fused with the gus gene were constructed and transformed into tobacco by Agrobacterium-mediated method, respectively. GUS expression in transgenic tobacco and activity analysis were conducted. The gus gene expression can be detected in the transgenic tobacco harboring each promoter segment, among which the driving activity of the full-length promoter 3f-P was the weakest, while the driving activity of the promoter segment 3f-P3 was the strongest. Upon exposure of the transgenic tobacco to salt stress, the GUS activity driven by 3f-P, 3f-P1, 3f-P2 and 3f-P3 was 3.3, 1.2, 1.9 and 1.2 times compared to that of the transgenic plants without salt treatment. This suggests that the AhRabG3f promoter was salt-inducible and there might be positive regulatory elements between 3f-P and 3f-P3 in response to salt stress. The results of GUS activity driven by promoter fragments after salt treatment showed that elements included MYB and GT1 between 1 930 bp and 1 745 bp. Moreover, a TC-rich repeat between 682 bp and 526 bp might be positive cis-elements responsible for salt stress, and an MYC element between 1 395 bp and 682 bp might be a negative cis-element responsible for salt stress. This study may facilitate using the induced promoter to regulate the salt resistance of peanut.
Arachis/genetics* ; Fabaceae/genetics* ; GTP-Binding Proteins/metabolism* ; Gene Expression Regulation, Plant ; Glucuronidase/metabolism* ; Plant Proteins/metabolism* ; Plants, Genetically Modified/genetics* ; Salt Stress ; Stress, Physiological/genetics* ; Tobacco/genetics*

Creating new germplasms and breeding new cultivars in peanut by radiation mutagenesis and tissue culture were conducted in this study, aiming to develop new breeding method of peanut. Mature seeds from Luhua 11, the most commonly grown peanut cultivar in Northern China, were treated by fast neutron irradiation. Then the embryo leaflets were separated from the irradiated seeds and inoculated on the media, and the regenerated seedlings were obtained through somatic embryogenesis pathway. The regenerated seedlings were grafted, acclimated and then transplanted into field and the selfed pods were harvested from 83 regenerated plants. The progenies were selected by the pedigree method, and 107 mutants were obtained from the progenies of the 83 regenerated plants. Different mutants showed obvious variation in many agronomic traits, including main stem height, branch number, pod shape and size, seed coat color, inner seed coat color, oil content and protein content etc. Yuhua 7, a new peanut variety with low oil content, early maturity and waterlogging tolerance was obtained. The yield of Yuhua 7 was over 14% higher than that of the mutagenic parent Luhua 11, and the oil content of kernels was 47.0%, lower than that of parent Luhua 11 with 52.1% oil content. Yuhua 7 had passed peanut variety regional multi-location trials in Liaoning Province in 2016 and its average yield was 13.8% higher than that of the control variety Baisha 1017. It had also passed national peanut variety registration, and the registration ID is "GPD peanut (2018) 370105". The results show that irradiation mutagenesis combined with tissue culture is an effective method for creating new germplasm and breeding new varieties of peanut.
Arachis ; Breeding ; China ; Fast Neutrons ; Plant Breeding ; Seeds

Leaf water potential of peanut subjected to drought stress is positively related to the oil content of peanut kernels. The aim of this study was to directly screen the high oil mutants of peanut and create the new peanut varieties using hydroxyproline as water potential regulator. In vitro mutagenesis was carried out with the embryonic leaflets of peanut variety Huayu 20 as explants and pingyangmycin as a mutagen added into the somatic embryo formation medium. The formed somatic embryos were successively transferred to somatic embryo germination and selection medium containing 6 mmol/L hydroxyproline (at -2.079 MPa water potential ) to induce regeneration and directionally screen high oil content mutants. After that, these plantlets were grafted and transplanted to the experimental field and 132 high oil mutants with oil content over 55% were obtained from the offspring of regenerated plants. Finally, among them, the oil contents of 27 lines were higher than 58% and of 2 lines were higher than 60%. A new peanut variety Yuhua 9 with high yield and oil content was bred from the regenerated plant progenies combining the pedigree breeding method. The yield was 14.0% higher than that of the control cultivar in the testing new peanut varieties of Liaoning province, and also it has passed the national registration of non-major crop varieties. Yuhua 9 with an oil content of 61.05%, which was 11.55 percentage points higher than that of the parent Huayu 20, was the peanut cultivar with the highest oil content in the world. The result showed that it was an effective way for directional breeding of high oil peanut varieties by means of the three-step technique including in vitro mutagenesis, directional screening by reducing water potential in medium and pedigree selection of regenerated plant progenies.
Arachis ; Droughts ; Germination ; Mutagenesis ; Plant Breeding

Objective: To study the response characteristics of the posterior intralaminar nucleus (PIN) of auditory thalamus in VGluT2-Cre transgenic adult mice when exposed to white noise and 10K pure tone stimulation. Methods: All adult male Vglut2-Cre mice (8-12 weeks) were used in this study between Oct, 2017 and Oct, 2018. Using the calcium signal fiber photometry method, optic fiber was employed to locate on PIN by injecting AAV-hSyn-DIO-GCaMP6m virus, and thereafter, the activity of the target cluster neurons during different acoustic stimuli was recorded. Matlab was used for data processing and statistical analysis. Results: (1)In both white noise and 10 kHz pure tone as a continuous three-second stimulation, the peak amplitude of calcium signal activity generated in PIN by white noise was superior to that of pure tone, the statistic result showed significantly difference (n=6, t=2.404, P=0.037 1) . (2)In addition, when white noise and 10K pure tone played as consecutive 3 or 5 pips within three-second stimulation, the stimulus-following ability in a consecutive 3 pulses play within 3 seconds was far better than a consecutive 5 pips play within 3 seconds (in both white noise and 10 kHz pure tone), yet consecutive 3 pips play showed greater signal attenuation speed than that in consecutive 5 pips play, the statistic result showed significantly difference (n=6, t=2.748 P=0.033 4) .(3)Regardless of the intra-group comparisons between white noise and 10 kHz pure tone stimulation, PIN showed better signal response in a consecutive 3 pips play than consecutive 5 pips play or a continuous three-second stimulation. When came to the statistical analysis, the acoustic response degree of a continuous three-second stimulation was an intermediate between two others, both consecutive 3 or 5 pips play showed significantly difference. Conclusions The results suggest that under the same acoustic intensity, VGluT2-Cre transgenic adult mice′s PIN shows greater signal response in white noise than pure tone. PIN shows greater signal attenuation to repetition play of 10 kHz pure tone, which implies PIN shows stronger adaptation to 10 kHz pure tone than to white noise. Lastly, PIN is more responsive to a complex sound information (white noise) than to simple sound information (pure tone).

AIM:To study the effect of roscovitine on the inflammatory hyperplasia of carotid artery intima in rats and the related mechanisms .METHODS: SD rats ( n=60 ) were randomly divided into 3 groups including control group, model group and treatment group .The rat model was established by trypsin digestion injury .The rats in control group were given sham operation .The rats in treatment group were administered with 0.5 mL roscovitine (2 g/L) slow-re-leasing gelatin.The rats in each group were fed normally for 4 weeks, then killed to take out carotid arteries for further ob-servations .The effects of roscovitine on the inflammatory hyperplasia of carotid artery intima and the related mechanism via nuclear factor-κB ( NF-κB) in the rats were detected by Western blot .RESUITS:Roscovitine inhibited the activation of NF-κB and the expression of inflammatory factors cyclooxygenase-2 (COX-2), vascular cell adhesion molecule-1 (VCAM-1),TNF-αand IL-6 via blocking the phosphorylation activation of NF-κB and inhibiting the degradation of IκB-α.CON-CLUSION:Roscovitine inhibits inflammatory hyperplasia of carotid artery intima in the rats via suppressing NF-κB activa-tion.

Cryptotanshinone (CPT) is a natural compound isolated from traditional Chinese medicine Salvia miltiorrhiza Bunge. In the present study, the regulatory effect and potential mechanisms of CPT on tumor necrosis factor alpha (TNF-α) induced lectin-like receptor for oxidized low density lipoprotein (LOX-1) were investigated. Human umbilical vein endothelial cells (HUVECs) were cultured and the effect of TNF-α on LOX-1 expression at mRNA and protein levels was determined by Real-time PCR and Western blotting respectively. The formation of intracellular ROS was determined with fluorescence probe CM-DCFH2-DA. The endothelial ox-LDL uptake was evaluated with DiI-ox-LDL. The effect of CPT on LOX-1 expression was also evaluated with SD rats. TNF-α induced LOX-1 expression in a dose- and time-dependent manner in endothelial cells. TNF-α induced ROS formation, phosphorylation of NF-κB p65 and ERK, and LOX-1 expression, which were suppressed by rotenone, DPI, NAC, and CPT. NF-κB inhibitor BAY11-7082 and ERK inhibitor PD98059 inhibited TNF-α-induced LOX-1 expression. CPT and NAC suppressed TNF-α-induced LOX-1 expression and phosphorylation of NF-κB p65 and ERK in rat aorta. These data suggested that TNF-α induced LOX-1 expression via ROS activated NF-κB/ERK pathway, which could be inhibited by CPT. This study provides new insights for the anti-atherosclerotic effect of CPT.
Animals ; Aorta ; Blotting, Western ; Endothelial Cells* ; Fluorescence ; Human Umbilical Vein Endothelial Cells ; Lipoproteins ; Medicine, Chinese Traditional ; Phosphorylation ; Rats ; Reactive Oxygen Species* ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Rotenone ; Salvia miltiorrhiza ; Tumor Necrosis Factor-alpha

Abstrac:Aim To study the effect of hydroxysafflor yellow A ( HYSA ) on the proliferation of vascular smooth muscle cells ( VSMCs) and the related molecu-lar mechanism. Methods The inhibitory effects of hydroxysafflor yellow A on VSMC proliferation was de-tected using cell culture, MTT assay, Western blot and immunohistochemical staining. Results The results showed that HYSA inhibited cell proliferation induced by PDGF in a dose-dependent (5,10,20,40 μmol· L-1 ) manner, reduced proliferating cell nuclear anti-gen ( PCNA ) expression and blocked PDGFR-MEK-ERK1/2 signaling pathway activated by PDGF in VSMCs. Conclusion HYSA inhibits VSMCs prolifer-ation via reducing the expression of PCNA and blocking signal transduction of MEK-ERK1/2 in VSMCs.