ObjectiveThis paper aims to investigate the material basis of the anti-inflammatory efficacy and mechanism of action of Bushen Tongdu prescription （BSTDP）. MethodsThe chemical components of BSTDP and its blood-absorbed components in vivo were systematically identified by using ultra-performance liquid chromatography-linear ion trap-electrostatic field orbitrap high-resolution mass spectrometry （UPLC-LIT-Orbitrap-MS）. Network pharmacology was employed to screen blood-absorbed bioactive components and potential targets of this formula. A protein-protein interaction （PPI） network of core targets was constructed to conduct enrichment analysis. Molecular docking was further utilized to verify the binding affinity between key components and targets. The inflammatory model was established and verified in vivo by using a transgenic zebrafish Tg （mpx： GFP）. At three days post-fertilization （3 dpf）， larvae of zebrafish were randomly assigned to blank group， model group， positive drug dexamethasone acetate group （75 μmol·L^-1）， and BSTDP groups with low， medium， and high doses （500， 1 000， and 2 000 mg·L^-1）. The distribution and quantity of neutrophils in the yolk sac region were observed under a fluorescence microscope. The mRNA expression levels of key genes in the toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor kappa-B （NF-κB） signaling pathway and inflammatory factors including interleukin （IL）-1β， IL-6， and tumor necrosis factor-α （TNF-α） were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsA total of 120 chemical components were identified in BSTDP， among which 26 original components were confirmed by using serum pharmacochemical methods. A total of 227 common targets linking rheumatoid arthritis （RA） and the blood-absorbed components were screened by network pharmacology. It is suggested that pseudobrucine， vomicine， sinapine， rehmannioside， cinnamyl alcohol glycoside， and methylephedrine exert anti-inflammatory effects by acting on core targets including protein kinase B1 （Akt1）， signal transducer and activator of transcription 3 （STAT3）， tumor necrosis factor （TNF）， TLR4， mitogen-activated protein kinase 14 （MAPK14）， and phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit α （PIK3CA）， thereby modulating multiple signaling pathways such as TLR4 and NF-κB. In vivo verification in zebrafish demonstrates that the maximum tolerable concentration of Bushen Tongdu Formula is 2 000 mg·L^-1. Compared to those in the blank group， zebrafish in the model group showed a significantly higher number of neutrophils in the yolk sac region （P<0.01） and rising mRNA levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β （P<0.01）. Compared to that in the model group， the number of neutrophils was significantly reduced in BSTDP groups with medium and high doses， as well as the dexamethasone acetate group （P<0.05， P<0.01）. There was no statistically significant difference in the low dose group. The mRNA expression levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β were significantly down-regulated （P<0.05， P<0.01）. ConclusionThis paper identifies the material basis of the efficacy of BSTDP， demonstrating that the formula can exert an anti-inflammatory effect through the TLR4/MyD88/NF-κB signaling pathway. The results provide scientific experimental evidence for its further clinical application.

ObjectiveThis paper aims to investigate the material basis of the anti-inflammatory efficacy and mechanism of action of Bushen Tongdu prescription （BSTDP）. MethodsThe chemical components of BSTDP and its blood-absorbed components in vivo were systematically identified by using ultra-performance liquid chromatography-linear ion trap-electrostatic field orbitrap high-resolution mass spectrometry （UPLC-LIT-Orbitrap-MS）. Network pharmacology was employed to screen blood-absorbed bioactive components and potential targets of this formula. A protein-protein interaction （PPI） network of core targets was constructed to conduct enrichment analysis. Molecular docking was further utilized to verify the binding affinity between key components and targets. The inflammatory model was established and verified in vivo by using a transgenic zebrafish Tg （mpx： GFP）. At three days post-fertilization （3 dpf）， larvae of zebrafish were randomly assigned to blank group， model group， positive drug dexamethasone acetate group （75 μmol·L^-1）， and BSTDP groups with low， medium， and high doses （500， 1 000， and 2 000 mg·L^-1）. The distribution and quantity of neutrophils in the yolk sac region were observed under a fluorescence microscope. The mRNA expression levels of key genes in the toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor kappa-B （NF-κB） signaling pathway and inflammatory factors including interleukin （IL）-1β， IL-6， and tumor necrosis factor-α （TNF-α） were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsA total of 120 chemical components were identified in BSTDP， among which 26 original components were confirmed by using serum pharmacochemical methods. A total of 227 common targets linking rheumatoid arthritis （RA） and the blood-absorbed components were screened by network pharmacology. It is suggested that pseudobrucine， vomicine， sinapine， rehmannioside， cinnamyl alcohol glycoside， and methylephedrine exert anti-inflammatory effects by acting on core targets including protein kinase B1 （Akt1）， signal transducer and activator of transcription 3 （STAT3）， tumor necrosis factor （TNF）， TLR4， mitogen-activated protein kinase 14 （MAPK14）， and phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit α （PIK3CA）， thereby modulating multiple signaling pathways such as TLR4 and NF-κB. In vivo verification in zebrafish demonstrates that the maximum tolerable concentration of Bushen Tongdu Formula is 2 000 mg·L^-1. Compared to those in the blank group， zebrafish in the model group showed a significantly higher number of neutrophils in the yolk sac region （P<0.01） and rising mRNA levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β （P<0.01）. Compared to that in the model group， the number of neutrophils was significantly reduced in BSTDP groups with medium and high doses， as well as the dexamethasone acetate group （P<0.05， P<0.01）. There was no statistically significant difference in the low dose group. The mRNA expression levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β were significantly down-regulated （P<0.05， P<0.01）. ConclusionThis paper identifies the material basis of the efficacy of BSTDP， demonstrating that the formula can exert an anti-inflammatory effect through the TLR4/MyD88/NF-κB signaling pathway. The results provide scientific experimental evidence for its further clinical application.

Objective To explore the differential expression genes(DEGs)and related signaling pathways of the Qi deficiency and blood stasis(QDBS)and Yin deficiency and blood stasis(YDBS)syndromes in ischemic stroke(IS)at the molecular level,elucidating the potential mechanisms and biological basis of blood stasis syndrome in IS.Methods The mRNA expression profile dataset GSE100235 of the two syndromes of IS rats was downloaded from the GEO database,and the limma package of R language was used to screen DEGs.Subsequently,GO and KEGG analyses were performed using the DAVID database.The miRDB and miRWalk databases were used to predict the miRNAs of DEGs,and aa miRNA-mRNA regulatory network was constructed.A protein-protein interaction network was built using the STRING database,and the cytoHubba plug-in was utilized to identify the core target genes.The rat model of QDBS in IS was established by exhaustive swimming training combined with modified Longa's thread embolism,and the rat model of YDBS in IS was established by hydrocortisone injection and thread embolism.RT-qPCR was employed to validate the expression levels of core target genes in rat brain tissues of the two groups.Results A total of 47 DEGs were identified.These genes were mainly involved in biological processes such as neuronal apoptosis,inflammatory response,and adaptive immune response,as well as pathways related to lipid metabolism,atherosclerosis,and C-type lectin receptor signaling.The miRNA-mRNA interaction network consisted of 64 nodes and 55 edges.Five core target genes were obtained,including Ccl2,Selp,Timp1,Ccr1l1,and Fpr1.Conclusion There are significantly differentially expressed genes in QDBS and YDBS syndrome of IS rats.These genes are involved in a variety of biological processes and pathways,and are most closely related to lipid metabolism and atherosclerosis signaling pathways.

Objective To explore the differential expression genes(DEGs)and related signaling pathways of the Qi deficiency and blood stasis(QDBS)and Yin deficiency and blood stasis(YDBS)syndromes in ischemic stroke(IS)at the molecular level,elucidating the potential mechanisms and biological basis of blood stasis syndrome in IS.Methods The mRNA expression profile dataset GSE100235 of the two syndromes of IS rats was downloaded from the GEO database,and the limma package of R language was used to screen DEGs.Subsequently,GO and KEGG analyses were performed using the DAVID database.The miRDB and miRWalk databases were used to predict the miRNAs of DEGs,and aa miRNA-mRNA regulatory network was constructed.A protein-protein interaction network was built using the STRING database,and the cytoHubba plug-in was utilized to identify the core target genes.The rat model of QDBS in IS was established by exhaustive swimming training combined with modified Longa's thread embolism,and the rat model of YDBS in IS was established by hydrocortisone injection and thread embolism.RT-qPCR was employed to validate the expression levels of core target genes in rat brain tissues of the two groups.Results A total of 47 DEGs were identified.These genes were mainly involved in biological processes such as neuronal apoptosis,inflammatory response,and adaptive immune response,as well as pathways related to lipid metabolism,atherosclerosis,and C-type lectin receptor signaling.The miRNA-mRNA interaction network consisted of 64 nodes and 55 edges.Five core target genes were obtained,including Ccl2,Selp,Timp1,Ccr1l1,and Fpr1.Conclusion There are significantly differentially expressed genes in QDBS and YDBS syndrome of IS rats.These genes are involved in a variety of biological processes and pathways,and are most closely related to lipid metabolism and atherosclerosis signaling pathways.

Objective:To investigate the role and mechanisms of fibulin-1 in senescence-related calcification of rat vascular smooth muscle cells induced by high-concentrationphosphate treatment.Methods:From September 2020 to September 2021, rat primary vascular smooth muscle cells were extracted from the thoracic aorta and abdominal aorta of 10 male SD rats aged 6 to 8 weeks.Phosphate(2.5 mmol/L Pi)was used to stimulate the calcification of vascular smooth muscle cells(VSMCs)in a model of stress-induced senescence-related calcification.Cellular senescence was assessed by SA-β-gal staining.Cellular calcification was determined by alizarin red staining and quantification of calcium deposition.Phenotypic transformation indexes and the expression of fibulin-1 during the process of calcification were detected by Western blot.The expression of fibulin-1 in primary rat vascular smooth muscle cells was knocked down by siRNA, the expression of pSmad3 was detected by immunofluorescence, and the effects of fibulin-1 on phenotypic transformation indexes of smooth muscle cells were detected by Western blot.The cells were cultured with recombinant fibulin-1 while transforming growth factor beta(TGF-β)inhibitor A83-01 and pSmad3 inhibitor SIS3 were also added.The senescence and calcification indexes of smooth muscle cells were detected by Western blot.Results:In the stress-induced aging model with phosphate stimulation of calcification in rat VSMCs, the expression of fibulin-1 was up-regulated( t=11.20, P<0.01), the expressions of MHC and SM22α was down-regulated( t=7.97, P<0.01; t=10.27, P<0.01), and the expression of osteoblastic phenotype markers OPN and Bmp2 and senescence marker P53 was up-regulated( t=4.79, P<0.01; t=9.56, P<0.01; t=14.07, P<0.01). Knockdown of fibulin-1 attenuated the degree of senescence and calcium deposition in VSMCs( t=12.90, P<0.05)and decreased the expression of OPN, Bmp2 and P53( t=5.92, P<0.05; t=10.15, P<0.01; t=8.28, P<0.01), at the same time, and TGF-β and pSmad3 expression was inhibited( t=12.90, P<0.01; t=7.46, P<0.01). After the addition of TGF-β/ smad3 pathway inhibitors, the stimulatory effect of recombinant fibulin-1 on phenotypic transformation and senescence protein expression inVSMCs was significantly reduced( t=4.52, P<0.01; t=9.82, P<0.01; t=3.85, P<0.05). Conclusions:Fibulin-1 can promote aging-related calcification of vascular smooth muscle cells through the TGF-β/smad3 signaling pathway.

Objective:To explore the differences of risk stratification of very high-risk or extreme high-risk atherosclerotic cardiovascular diseases (ASCVD) and the attainment rates of low-density lipoprotein cholesterol (LDL-C) management targets evaluated by three different criteria, and the causal attributions of these differences.Methods:Patients with ASCVD were consecutively enrolled from January 1 to December 31 in 2019, and were evaluated for very high-risk or extreme high-risk and LDL-C goal attainment rates with 2018 American guideline on the management of blood cholesterol (2018AG), 2019 China Cholesterol Education Program (CCEP) Expert Advice for the management of dyslipidemias (2019EA) and 2020 Chinese expert consensus on lipid management of very high-risk ASCVD patients(2020EC), respectively. The causal attributions of the differences in attainment rates were analyzed as well.Results:A total of 1 864 ASCVD patients were included in this study. According to 2018AG, 2019EA and 2020EC, the proportions of the patients with very high-risk or extreme high-risk were 59.4%, 90.7%, and 65.6%, respectively. The absolute LDL-C target attainment rates were 37.2%, 15.7%, and 13.7%, respectively, the differences between each two rates were statistically significant (all P<0.001). As to the differences in attainment rates between 2020EC and 2018AG, 61.5% were due to the different LDL-C goal attainment values and 38.5% were caused by the different risk stratifications, while for the differences between 2020EC and 2019EA attainment rates, different LDL-C goal attainment values were responsible for 13.2%, and different risk stratifications were responsible for 86.8% of the differences. Conclusions:There are significant differences in the proportions and LDL-C attainment rates among the three different criteria for very high-risk or extreme high-risk ASCVD. 2020EC showed a moderate proportion of patients with extreme high-risk, and had the lowest LDL-C attainment rate. The differences between 2020EC and 2018AG are mainly due to the LDL-C target values, and the differences between 2020EC and 2019EA are mainly caused by the risk stratifications.

Objective:To evaluate the adjuvant role of the eCura scoring system in selecting appropriate treatment strategies after non-curative endoscopic submucosal dissection (ESD) of early gastric cancer (EGC) patients.Methods:The clinicopathological data of 110 EGC patients who underwent non-curative ESD at Fujian Provincial Hospital from January 2015 to June 2019 were retrospectively analyzed. According to the eCura score, patients were divided into three lymph node metastasis (LNM) risk groups: low-risk group (79 cases), middle-risk group (22 cases), and high-risk group (9 cases). The receiver operator characteristic (ROC) curve analysis was used to test the diagnostic efficacy of eCura scoring system in predicting LNM. Logistic regression analysis was used to explore the influence of risk stratification of eCura scoring system on LNM. Kaplan-Meier method was used to evaluate cancer survival rate, which was then compared with log-rank test.Results:Thirty-five patients underwent additional standard surgery after ESD, including 22 in the low-risk group, 8 in the middle-risk group, and 5 in the high-risk group. Among them, 5 cases had LNM, including 1 case in the low-risk group and the middle-risk group respectively and 3 cases in the high-risk group. The area under the ROC curve was 0.857 (95% CI: 0.697-0.952, P=0.001), and when the cut-off value of the eCura score was set at 3, the Yuden index reached the maximum value of 0.7, with the corresponding sensitivity and specificity of 80% and 90%, respectively. Logistic regression analysis showed that the probability of LNM in the middle-risk group was about 3.00 times (95% CI: 0.17-54.57, P=0.458) as high as that in the low-risk group, and the probability of LNM in the high-risk group was about 31.50 times (95% CI: 2.14-463.14, P=0.012) of that in the low-risk group. The follow-up time was 12 to 58 months, and the median follow-up time was 40 months. There were 10 cases of recurrence, including 4 cases in the low-risk group, 3 cases in the middle-risk group and 3 cases in the high-risk group, of which 2 cases in the low-risk group were from those of additional standard surgery after ESD, and the remaining 8 cases were from those who did not receive additional standard surgery after ESD. Kaplan-Meier survival curve analysis showed that the survival rate of patients with additional surgery in the low-risk group was similar to that of patients without ( P=0.319), and the survival rate of patients with additional surgery in the middle-risk group was also similar to that of patients without ( P=0.296). The survival rate of patients with additional surgery in the high-risk group was significantly higher than that of those without ( P=0.013). Conclusion:The eCura scoring system can assist the selection of treatment strategies after non-curative resection of EGC, and can accurately predict the risk of subsequent LNM and recurrence. Close follow-up may be an acceptable option for patients with low risk of LNM, and additional standard surgical treatment may be more conducive to improving the prognosis in patients with high risk of LNM.

Objective:To evaluate type B2 vessels for predicting the invasion depth of superficial esophageal squamous cell carcinoma (SESCC) under narrow band imaging-magnifying endoscopy (NBI-ME), and to analyse the influencing factors of over-prediction and under-prediction.Methods:A total of 86 SESCC patients with 86 lesions confirmed by postoperative pathology, who underwent NBI-ME to evaluate the invasion depth of esophageal tumor and were observed for type B2 vessels in Department of Digestive Endoscopy of Fujian Provincial Hospital from January 2015 to April 2020, were included in the case-control study. According to the postoperative pathological results, the patients were divided into the correct prediction group ( n=25) and the wrong prediction group ( n=61), and the accuracy of prediction was calculated. The wrong prediction group was further divided into the over-prediction group ( n=49) and the under-prediction group ( n=12) to find the influencing factors of over-prediction and under-prediction, respectively. Further, multivariate Logistic analysis was performed to explore the independent influential factors for the prediction. Results:The accuracy of type B2 vessels under NBI-ME for predicting the invasion depth of SESCC was only 29.07% (25/86), and the over-prediction rate and under-prediction rates were 56.98% (49/86) and 13.95% (12/86), respectively. Univariate analysis showed that the classification of type B2 area ( χ2=36.25, P<0.001), the distinct endoscopic features (nodules, thickening and obvious depression, χ2=22.90, P<0.001), and inflammation around type B2 vessels ( χ2=9.54, P=0.004) were related to the over-prediction of the invasion depth of SESCC, and the distinct endoscopic features were related to the under-prediction of the invasion depth ( P=0.016). Multivariate Logistic regression analysis showed that B2-narrow (type B2 area ≤5 mm) ( P<0.001, OR=241.988,95% CI:15.229-3 845.252) and inflammation around type B2 vessels ( P=0.033, OR=12.801, 95% CI: 1.226-133.713) were independent risk factors for over-predicting the invasion depth of SESCC, while the distinct endoscopic features were independent protective factors for over-prediction ( P<0.001, OR=0.012, 95% CI: 0.001-0.150). The distinct endoscopic feature was an independent risk factor for under-predicting invasion depth of SESCC with type B2 vessels ( P=0.027, OR=7.899, 95% CI: 1.259-49.565). Conclusion:The accuracy of predicting the invasion depth of SESCC is low only based on type B2 vessels in NBI-ME, and over-prediction is prone to occur. B2-narrow, inflammation around type B2 vessels and without nodules, thickening and obvious depression are closely related to the over-prediction of invasion depth of SESCC by type B2 in NBI-ME, and nodules, thickening and obvious depression are closely associated with the under-prediction. However, whether the combined assessment of type B2 vessels under NBI-ME and endoscopic manifestation can improve the accuracy of prediction needs to be confirmed by further studies.

Objective:To analyze the composition and functional characteristics of the intestinal microflora in novel coronavirus pneumonia patients with arterial stiffness, in order to provide empirical evidence for rational use of intestinal microecological modulators.Methods:Patients with novel coronavirus pneumonia admitted to our hospital between April 5 and April 19, 2020 were enrolled as research subjects.They were divided into the stiffness group and the control group according to the results of arterial stiffness.Stool samples were collected within 7 days of admission.Intestinal flora DNA was analyzed and entered into a database, shotgun metagenomic sequencing was performed, and bioinformatics analysis was conducted based on sequencing results.Results:A total of 16 patients with novel coronavirus pneumonia were included in this study, including 7 in the stiffness group and 9 in the control group.Brachial-ankle pulse wave velocity and blood pressure were higher in the stiffness group than in the control group( P<0.05). Beta diversity analysis at the phylum level showed that there were significant differences in the composition of the intestinal flora between the two groups( P<0.05). Patients with arterial stiffness had a lower relative abundance than the controls in fecal Holdemanella, Mitsuokella, Deinococcus, Lachnospira, Turicibacter, Butyrivibrio, Sporomusa, and Halanaerobium, species associated with the production of short-chain fatty acids(SCFAs), regulation of energy metabolism, anti-radiation, anti-oxidative stress and anti-inflammatory effects.The Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis showed that the intestinal flora in the control group was mainly involved in the pathways of lipopolysaccharide biosynthesis, metabolism of SCFA and other amino acids, and membrane transport, while the intestinal flora in the stiffness group was mostly concerned with the pathways of amino acid metabolism and DNA damage repair. Conclusions:The use of microbial agents capable of increasing short-chain fatty acids in patients with novel caronavirus infection may contribute to the restoration of intestinal flora homeostasis.

Sterol-regulatory element binding proteins (SREBPs) are the key transcriptional regulators of lipid metabolism. The activation of SREBP requires translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi, where it is sequentially cleaved by site-1 protease (S1P) and site-2 protease and releases a nuclear form to modulate gene expression. To search for new genes regulating cholesterol metabolism, we perform a genome-wide CRISPR/Cas9 knockout screen and find that partner of site-1 protease (POST1), encoded by C12ORF49, is critically involved in the SREBP signaling. Ablation of POST1 decreases the generation of nuclear SREBP and reduces the expression of SREBP target genes. POST1 binds S1P, which is synthesized as an inactive protease (form A) and becomes fully mature via a two-step autocatalytic process involving forms B'/B and C'/C. POST1 promotes the generation of the functional S1P-C'/C from S1P-B'/B (canonical cleavage) and, notably, from S1P-A directly (non-canonical cleavage) as well. This POST1-mediated S1P activation is also essential for the cleavages of other S1P substrates including ATF6, CREB3 family members and the α/β-subunit precursor of N-acetylglucosamine-1-phosphotransferase. Together, we demonstrate that POST1 is a cofactor controlling S1P maturation and plays important roles in lipid homeostasis, unfolded protein response, lipoprotein metabolism and lysosome biogenesis.