To investigate chemical constituents contained in cytotoxic petroleum ether extractive fractions from ethanol extracts of Cirsium setosum. The constituents were separated and purified by a combination of various chromatographic methods including silica gel, Sephadex LH-20, and preparative HPLC. Structures of the isolates were elucidated by spectroscopic methods including 1D, 2D NMR and MS methods. The compound structures were also determined by reference to literature. Twelve compounds were separated from the petroleum ether fraction of ethanolic extract and elucidated as lupenyl acetate (1), lupeol (2), lupenone (3), beta-amyrin (4), psi-taraxasterol (5), psi-taraxasteryl acetate (6), taraxasteryl acetate (7), marsformoxide B (8), alpha-amyrenone (9), beta-amyrenone (10), taraxasterone (11) and psi-taraxasterone (12). Of them, compounds 3, 5, 7-12 were separated from this genus for the first time.
Chromatography, High Pressure Liquid ; Cirsium ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Magnetic Resonance Spectroscopy ; Oleanolic Acid ; analogs & derivatives ; chemistry ; Pentacyclic Triterpenes ; chemistry ; Sterols ; chemistry ; Triterpenes ; chemistry

Objective:To investigate the stability and feasibility of improved silent MRA technique based on hybrid-arterial spin labeling(ASL) for imaging intracranial arterial stenosis.Methods:From September 2019 to May 2020, totally 35 patients with suspected intracranial vascular stenosis in Department of Neurology of Northern Jiangsu People′s Hospital were enrolled in this study. Silent MRA and improved silent MRA based on hybrid-ASL technique were performed respectively. The acquisition noise (noise measurement and subjective score) of two kinds of MRA examination were evaluated respectively. Two neuroradiologists performed image quality scoring and signal-to-noise ratio (SNR) measurement of intracranial arteries (including internal carotid artery, vertebrobasilar artery, anterior cerebral artery, middle cerebral artery, and posterior cerebral artery) in the two kinds of MRA images using a double-blind, completely randomized method. Independent sample t-test was used to compare the image quality and SNR of two kinds of MRA images in each segment. Two experts assessed the degree of stenosis at the site of confirmed intracranial artery stenosis. Kappa test was used to assess interobserver and intermodel agreement. Results:There was no significant difference in acquisition noise between improved silent MRA and silent MRA ( P>0.05). In all five segments measured, the image quality scores of internal carotid artery [(4.40±0.49)scores], anterior cerebral artery[(4.30±0.33)scores] and middle cerebral artery [(4.46±0.34)scores] in improved silent MRA were higher than those in silent MRA images [(4.02±0.43)scores, (4.02±0.31)scores, (4.02±0.31)scores; t=2.825, 2.877, 1.683, all P<0.05)]. The SNR of internal carotid artery (9.11±1.23) and middle cerebral artery (8.77±1.87) in improved silent MRA images was higher than that in silent MRA images (7.83±1.33, 8.06±2.67, respectively; t=11.154, 3.268, both P<0.05). A total of 24 patients (38 lesions) with intracranial vascular stenosis were diagnosed by CTA. Improved silent MRA (Kappa=0.89, 95%CI 0.82-0.95) and silent MRA (Kappa=0.85, 95%CI 0.77-0.92) were highly consistent among observers in evaluating the degree of cerebrovascular stenosis.The results of improved silent MRA were highly consistent with those of CTA (Kappa=0.92, 95%CI 0.87-0.98), and those of silent MRA were highly consistent with those of CTA (Kappa=0.85, 95%CI 0.77-0.92). Conclusions:The improved silent MRA is feasible to improve the imaging quality and signal uniformity through efficient marking based on keeping the low noise features. In the diagnosis of intracranial stenosis and occlusive disease, the stability of improved silent MRA imaging improves the diagnostic efficiency of stenosis to a certain extent.

Objective:To analyze the anti-drug resistance, molecular epidemiology and virulence gene distribution of non-A-F group serotype isolates of Salmonella enterica enteric subspecis, so as to provide epidemiological basis for clinical diagnosis and treatment.Methods:Serotyping, antimicrobial susceptibility test and whole genome sequencing were performed on 11 isolates of non-A-F group serotype isolates of Salmonella enterica enteric subspecies that were isolated from The Children′s Hospital, Zhejiang University School of Medicine between 2017 and 2020. The serotype, multilocus sequence typing and virulence gene of the whole genome sequencing results were analyzed. Results:In our hospital, the detection rate of non-A-F group serotype isolates of Salmonella enterica enteric subspecies was low (1.13%). Among the 11 strains, there were 3 strains belong to Jangwani serotype, 2 strains of Hvittingfoss serotype, and Wandsworth, Pomona, Kedougou, Urbana, Poona and Kumasi serotypes have 1 strain each. Except for the two multi-drug resistant strains, the other strains were sensitive to most antibiotics, and the MICs were at low levels. A total of 9 ST types were detected in the 11 strains, the 3 Jangwani serotype strains were ST3918, and the other isolates were of different ST types. The phylogenetic tree shown that the three strains of Jangwani serotype were closely related. A total of 103 virulence genes were detected in the 11 strains, including 78 genes related to secretion system, 21 genes related to adherence, 2 genes related to magnesium uptake, 1 gene related to resistance to antimicrobial peptides and 1 gene related to typhoid toxin. Conclusions:The detection rate of the non-A-F group serotype isolates of Salmonella enterica enteric subspecies was low, and the sensitivity of the isolates to common antibiotics was high. The ST types and genetic relationship showed diversity. Clinical laboratory should pay attention to the detection of the non-A-F group serotype isolates of Salmonella enterica enteric subspecies, and the changes in drug resistance and virulence genes of the isolates should be closely monitored.

Objective:To clarify the effect and the mechanism of G protein-coupled receptor class C group 5 member A (GPRC5A) on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (GFs), thus to provide a foundation for delving into the role of G protein coupled receptor (GPCR) in periodontitis.Methods:Gingival tissue samples were collected from 3 individuals periodontally healthy (health group) and 3 patients with periodontitis (periodontitis group) in Shandong Stomatological Hospital from December 2022 to February 2023. The expressions of GPRC5A of the two groups were detected by immunohistochemistry staining. GFs used in this study were isolated from a portion of gingiva for the extraction of impacted teeth in School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University from December 2022 to February 2023. GFs were isolated with enzymic digestion and transfected with 30, 50 and 80 μmol/L small interfering RNA-GPRC5A (siGPRC5A) or small interfering RNA-negative control (siNC), regarded as the experimental group and the negative control one, respectively. The silencing efficiency of siGPRC5A was evaluated by real-time fluorescence quantitative PCR (RT-qPCR). Experiments were then conducted using these cells which were divided into four groups of negative control (NC), LPS, siGPRC5A+LPS and siGPRC5A. The mRNA and protein levels of GPRC5A in GFs under 1 mg/L LPS-induced GFs inflammatory state were evaluated by RT-qPCR and Western blotting analysis after GPRC5A knockdown. RT-qPCR was used to detect the gene expression levels of the inflammatory cytokines in GFs induced by LPS, namely, interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, prostaglandin endoperoxide synthase 2 (PTGS2) after GPRC5A knockdown. Western blotting analysis and immunofluorescence staining were used to further investigate the activation of nuclear factor-kappa B (NF-κB) signaling pathway.Results:Immunohistochemistry staining showed that the expression of GPRC5A in gingival tissues of periodontitis group (0.132±0.006) increased compared with that in periodontally healthy group (0.036±0.019) ( t=8.24, P=0.001). Meanwhile, RT-qPCR results showed that the gene expression levels of GPRC5A at different time point (2, 6, 12, 24 h) in LPS-induced GFs (0.026±0.002, 0.042±0.005, 0.004±0.000, 0.016±0.000) were upregulated compared with those in the NC group (0.004±0.000, 0.004±0.000, 0.002±0.000, 0.007±0.000) (all P<0.001), respectively, and peaked at 6 h. The 50 μmol/L group displayed the most significant decrease in siGPRC5A expression (31.16±3.29) compared with that of the siNC group (100.00±4.88) ( F=297.98, P<0.001). The results of RT-qPCR and Western blotting analysis showed that siGPRC5A (0.27±0.03, 0.71±0.00) suppressed the expressions of GPRC5A at both gene and protein levels, while LPS (1.30±0.10, 1.43±0.03) was able to promote the expressions of GPRC5A compared with those of the NC group (1.00±0.01, 1.00±0.00)(all P<0.001). The siGPRC5A+LPS group (0.39±0.03, 1.06±0.16) also inhibited the increase of GPRC5A at both gene and protein levels induced by LPS (1.30±0.10, 1.43±0.03) ( F=208.38, P<0.001; F=42.04, P<0.001). RT-qPCR results showed that the expressions of IL-8, IL-1β, IL-6, TNF-α, and PTGS2 at the gene level in LPS group were highly increased compared with those in the NC group (all P<0.001). siGPRC5A significantly suppressed LPS-induced expressions of these inflammatory cytokines in GFs (all P<0.001). Western blotting analysis showed that the levels of p65 and IκBα protein phosphorylation in the LPS group were highly increased compared with those in the NC group, and siGPRC5A could effectively suppressed LPS-induced protein phosphorylation (all P<0.01). Furthermore, immunofluorescence staining showed that NF-κB p65 in the control group was mainly concentrated in the cytoplasm, and partially translocated to the nucleus under the stimulation of LPS. siGPRC5A was able to inhibit LPS-induced intranuclear translocation of p65 to a certain extent. Conclusions:GPRC5A expression was upregulated in periodontitis, and GPRC5A knockdown inhibited LPS-induced inflammation. Moreover, GPRC5A played a role in inflammation regulation by interacting with NF-κB signaling pathway.