ObjectiveTo explore the possible mechanism of Yigan Fupi Prescription （抑肝扶脾方， YFP） in treating diarrhea-type irritable bowel syndrome （IBS-D） by investigating the AKT/mTOR signaling pathway. MethodsSixty SD rats were randomly divided into control group， model group， YFP low-， medium-， and high-dose group， and pinaverium bromide group， with 10 rats in each group. All groups but the control group， were subjected to 21 days of tail-clamping stimulation and 14 days of senna leaf gavage to establish a liver stagnation and spleen deficiency-type IBS-D rat model. After successful modeling， the YFP low-， medium-， and high-dose group were administered 0.96， 1.93， and 3.87 g/（kg·d） of the prescription， respectively. The pinaverium bromide group was given 13.5 mg/（kg·d）， while the control and model groups were given 10 ml/（kg·d） distilled water. All groups were administered once daily for 14 consecutive days. General conditions of the rats were recorded during the experiment， and after modeling and drug administration， body weight， Bristol stool score， abdominal withdrawal reflex （AWR） score， and histo pathology of colon tissue were observed under HE staining. ELISA was used to detect serum levels of tumor necrosis factor α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6）. Immunofluorescence was employed to detect the levels of AKT/mTOR pathway-related proteins including phosphorylated AKT （p-AKT）/AKT and phosphorylated mTOR （p-mTOR）/mTOR in the colon tissue. Western Blotting was used to detect the levels of autophagy-related proteins， including UNC-51-like kinase 1 （ULK1）， Beclin1 and LC3， and tight junction proteins including Occludin and ZO-1 in the colon tissue. ResultsAfter modeling， compared to the control group， the body weight of rats in the other groups decreased， and Bristol stool scores， as well as AWR scores under 20， 40， 60， and 80 mmHg increased （P＜0.05 or P＜0.01）. After drug administration， compared to the control group， the model group showed reduced body weight， decreased ULK1， Beclin1， LC3Ⅱ/LC3Ⅰ， Occludin， and ZO-1 protein levels in the colon tissue （P＜0.05 or P＜0.01）， and increased Bristol stool scores， AWR scores， serum TNF-α， IL-1β， and IL-6 levels， as well as p-AKT/AKT and p-mTOR/mTOR protein relative expression levels （P＜0.05 or P＜0.01）. Pathological results showed a significant reduction in goblet cells in the upper part of the glandular layer of the colon， with mild inflammatory cell infiltration. The submucosal collagen fibers were dissolved， with unclear boundaries， pale staining， and microvascular congestion and dilation. Compared with the model group， the YFP low-， medium-， and high-dose group and the pinaverium bromide group showed increased body weight， Beclin1， Occludin， and LC3Ⅱ/LC3Ⅰ protein levels （P＜0.05 or P＜0.01）， and decreased Bristol stool scores， AWR scores under 40， 60， and 80 mmHg， serum IL-1β， IL-6， TNF-α levels， and p-AKT/AKT， p-mTOR/mTOR protein relative expression levels （P＜0.05 or P＜0.01）. The pathological morphology of the rats in the YFP groups and pinaverium bromide group showed varying degrees of improvement. Compared with the pinaverium bromide group， the YFP low- and medium-dose group showed increased AWR scores under 20， 40， and 60 mmHg （P＜0.05）. The YFP low-dose group had reduced TNF-α， IL-1β， and IL-6 levels， and increased p-mTOR/mTOR protein relative expression levels occured in all YFP groups （P＜0.05）. Compared with the YFP low-dose group， the YFP high-dose group and pinaverium bromide group showed decreased AWR scores under different pressure levels and reduced p-AKT/AKT protein relative expression levels， while the YFP medium- and high-dose group had elevated serum TNF-α， IL-1β levels and reduced p-mTOR/mTOR protein relative expression levels （P＜0.05）. ConclusionYFP can effectively improve the pathological injury of colon tissue in IBS-D model rats with liver stagnation and spleen deficiency， reduce Bristol stool and AWR scores， and its mechanism may be related to reducing level of inflammatory factors and inhibiting AKT/mTOR pathway-related proteins in colon tissue， thereby enhancing the expression of autophagy-related proteins in the colon tissue.

OBJECTIVE To screen the potential active components of Polygonum orientale flower against myocardial ischemia- reperfusion injury （MIRI） in rats based on physiological and pathological states. METHODS SD rats were divided into normal control group， normal administration group， MIRI control group and MIRI administration group， with 5 rats in each group. After drug intervention or modeling and drug intervention， chromatographic separation plasma samples were collected， and chromatographic separation and mass spectrometry data collection were performed by using UPLC-Q-TOF/MS. The prototype components and metabolites were analyzed by comparing the reference substance maps， the maps of each plasma sample， and the relevant literature. At the same time， the common peaks in plasma samples of rats in normal administration group and MIRI administration group were identified. Combined with principal component analysis and orthogonal partial least square-discriminant analysis， the differential transitional components were screened out according to the value of variable importance in the projection （VIP）＞1， to speculate the potential active components of P. orientale flower in rats under physiological and pathological states. The SD rats were divided into control group， MIRI group， positive control group （Compound danshen tablets 0.2 g/kg， 3 times a day）， and potentially active compound groups （10 mg/kg， twice a day）， with 5 rats in each group. The rats in administration groups were given relevant medicine intragastrically， for 3 consecutive days. The activity of superoxide dismutase （SOD）， the leakages of lactate dehydrogenase （LDH）， creatine kinase isoenzyme-MB （CK-MB） and cardiac troponin Ⅰ （cTnⅠ） in plasma were detected after the last administration. RESULTS Twenty-six main chromatographic peaks were obtained from the total ion chromatogram of the extract of P. orientale flower， and 14 of them were determined， including gallic acid， catechin， protocatechuic acid and so on. There were fifteen （including 6 absorbed prototype components and 9 metabolites） and nineteen transitional components （including 6 absorbed prototype components and 13 metabolites） in the plasma sample of normal rats and MIRI rats. Eight transitional components were detected in both normal rats and MIRI rats， and the VIP values of kaempferol glucuronidation metabolites， quercetin carbonylation metabolites and N-p-paprazine to the corresponding peak were higher than 1. Compared with MIRI group， the activities of SOD were increased significantly in the plasma of MIRI rats in each potential active compound group （P＜0.01）， and the leakages of LDH， CK-MB， and cTnⅠ in the plasma of MIRI rats were reduced significantly （P＜0.01）. CONCLUSIONS The potential anti-MIRI active components in extract of P. orientale flower are N-p-paprazine， quercetin， kaempferol and kaempferol-3-O-β-D-glucoside.

Objective:To investigate the effects of carbon black and cadmium (Cd) combined exposure on autophagy and inflammatory response mediated by protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in human bronchial epithelial (16HBE) cells.Methods:In January 2022, human bronchial epithelial (16HBE) cells were resuscitated and cultured. Carbon black nanoparticles (CBNPs) were oxidized to adsorb Cd ions to construct "CBNPs-Cd" complexes. CCK-8 assay was used to detect the effects of different concentrations and time combinations of CBNPs and Cd on the viability of 16HBE cells. The subsequent dose groups were exposed to 2 μg/ml Cd, 100 μg/ml CBNPs, 100 μg/ml CBNPs+2 μg/ml Cd for 24 h. The number of autophagosomes and autolysosomes was detected by transmission electron microscopy. Western blotting was used to detect the protein expressions of PERK, eukaryotic initiation factor 2α (eIf2α), activating transcription factor 4 (ATF4), sequestosome 1 (SQSTM1/P62), and microtubule-associated protein 1 light chain 3 (LC3). After PERK gene was silenced by siRNA technology, the changes of autophagy marker proteins P62 and LC3 were detected, and the expressions of inflammatory factors interleukin-6 (IL6) and interleukin-8 (IL8) were detected by fluorescence quantitative PCR technique. One-way ANOVA analysis was used to compare three groups or more. LSD test was used for comparison between two groups. Factorial analysis was used for multivariate component analysis. Results:There was no significant change in cell viability of 16HBE after 24 h exposure to CBNPs and Cd alone or combined ( P>0.05). Compared with the control group, the expressions of P62 and LC3 in 16HBE cells were significantly increased in the CBNPs and Cd alone/combined exposure group ( P<0.05), and the number of autophagosomes and autophagolysosomes in the combined exposure group was increased compared with other groups. Compared with the control group, CBNPs and Cd alone exposure group had no significant effects on p-PERK/PERK and p-eIf2α/eIf2α protein expression ( P>0.05). However, the protein expressions of p-PERK/PERK and p-eIf2α/eIf2α and ATF4 were all increased in the combined exposure group ( P<0.05), and the levels of IL6 and IL8 in 16HBE cells in the combined exposure group of CBNPs and Cd were significantly higher than those in the control group ( P<0.05). The levels of LC3 protein, IL6 and IL8 were decreased in the CBNPs-Cd combined exposure group after knockdown of PERK gene ( P<0.05). The results of factorial analysis showed that exposure to CBNPs and Cd had significant effects on the expression of P62, LC3 and IL6 ( P<0.05), but the interaction between the two chemicals had no statistical significance ( P>0.05) . Conclusion:CBNPs-Cd combined exposure may inhibit autophagy and increase inflammation in human bronchial epithelial cells through activation of PERK-eIf2α-ATF4 pathway.

Objective:To investigate the effects of carbon black and cadmium (Cd) combined exposure on autophagy and inflammatory response mediated by protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in human bronchial epithelial (16HBE) cells.Methods:In January 2022, human bronchial epithelial (16HBE) cells were resuscitated and cultured. Carbon black nanoparticles (CBNPs) were oxidized to adsorb Cd ions to construct "CBNPs-Cd" complexes. CCK-8 assay was used to detect the effects of different concentrations and time combinations of CBNPs and Cd on the viability of 16HBE cells. The subsequent dose groups were exposed to 2 μg/ml Cd, 100 μg/ml CBNPs, 100 μg/ml CBNPs+2 μg/ml Cd for 24 h. The number of autophagosomes and autolysosomes was detected by transmission electron microscopy. Western blotting was used to detect the protein expressions of PERK, eukaryotic initiation factor 2α (eIf2α), activating transcription factor 4 (ATF4), sequestosome 1 (SQSTM1/P62), and microtubule-associated protein 1 light chain 3 (LC3). After PERK gene was silenced by siRNA technology, the changes of autophagy marker proteins P62 and LC3 were detected, and the expressions of inflammatory factors interleukin-6 (IL6) and interleukin-8 (IL8) were detected by fluorescence quantitative PCR technique. One-way ANOVA analysis was used to compare three groups or more. LSD test was used for comparison between two groups. Factorial analysis was used for multivariate component analysis. Results:There was no significant change in cell viability of 16HBE after 24 h exposure to CBNPs and Cd alone or combined ( P>0.05). Compared with the control group, the expressions of P62 and LC3 in 16HBE cells were significantly increased in the CBNPs and Cd alone/combined exposure group ( P<0.05), and the number of autophagosomes and autophagolysosomes in the combined exposure group was increased compared with other groups. Compared with the control group, CBNPs and Cd alone exposure group had no significant effects on p-PERK/PERK and p-eIf2α/eIf2α protein expression ( P>0.05). However, the protein expressions of p-PERK/PERK and p-eIf2α/eIf2α and ATF4 were all increased in the combined exposure group ( P<0.05), and the levels of IL6 and IL8 in 16HBE cells in the combined exposure group of CBNPs and Cd were significantly higher than those in the control group ( P<0.05). The levels of LC3 protein, IL6 and IL8 were decreased in the CBNPs-Cd combined exposure group after knockdown of PERK gene ( P<0.05). The results of factorial analysis showed that exposure to CBNPs and Cd had significant effects on the expression of P62, LC3 and IL6 ( P<0.05), but the interaction between the two chemicals had no statistical significance ( P>0.05) . Conclusion:CBNPs-Cd combined exposure may inhibit autophagy and increase inflammation in human bronchial epithelial cells through activation of PERK-eIf2α-ATF4 pathway.

Objective:To explore the toxic effect of carbon black nanoparticles on human bronchial epithelial cells, and identify the differentially expressed circular RNA based on the full transcriptome high-throughput sequencing, so as to provide evidence for the development of biomarkers exposed to carbon black nanoparticles and their application on epigenetic toxicology.Methods:In June 2020, 16 HBE cells were treated with carbon black nanoparticles at concentrations of 20, 40 and 80 μg/ml, and 16 HBE cells without any intervention were used as the control group. The cytotoxicity of carbon black nanoparticles was detected by CCK8 and LDH experiments. Real-time quantitative fluorescent PCR (qRT-PCR) and ELISA were used to detect the changes of interleukin-6 (IL-6) and interleukin-8 (IL-6, IL-8) mRNA and protein levels of carbon black nanoparticles with concentration gradient after 72 h exposure. Western blot analysis was conducted to detect the expression levels of toll-like receptor 4 (TLR4), phosphorylated nuclear factor-κB (P-NF-κB), apoptosis-related speckled protein (ASC) and Caspase-1 associated with nuclear factor-κB. According to high-throughput sequencing results, differentially expressed Circrnas were screened and identified by qRT-PCR, and those with stable differentially expressed circrnas and the strongest association with the NF-κB pathway were selected for ring performance identification.Results:After being exposed to carbon black nanoparticles for 72 h, the activity of 16HBE cells decreased significantly ( P<0.05), and the release of lactate dehydrogenase increased significantly ( P<0.05). Compared with control group, mRNA expression levels of IL-6 and IL-8, protein levels of IL-6 and IL-8 were increased, and protein levels of TLR4, p-NF-κB, ASC and Caspase-1 were significantly up-regulated in 16 HBE cells of different concentrations, with statistical significance ( P<0.05). Compared with the control group, a total of 492 differentially expressed circular Rnas (|log2 FC|>1) were detected. Among the 5 differentially expressed ( P<0.05) circular Rnas, circ_002642 was selected as the object of subsequent research on circular Rnas, affter 72 hours of exposure to 80 μg/ml CBNPs, 16HBE cells showed signlficantly higher expression of circ_002642 ( P<0.05) . Conclusion:Carbon black nanoparticles can induce differentially expressed circular RNAs associated with inflammatory response in human bronchial epithelial cells.

Objective:To explore the toxic effect of carbon black nanoparticles on human bronchial epithelial cells, and identify the differentially expressed circular RNA based on the full transcriptome high-throughput sequencing, so as to provide evidence for the development of biomarkers exposed to carbon black nanoparticles and their application on epigenetic toxicology.Methods:In June 2020, 16 HBE cells were treated with carbon black nanoparticles at concentrations of 20, 40 and 80 μg/ml, and 16 HBE cells without any intervention were used as the control group. The cytotoxicity of carbon black nanoparticles was detected by CCK8 and LDH experiments. Real-time quantitative fluorescent PCR (qRT-PCR) and ELISA were used to detect the changes of interleukin-6 (IL-6) and interleukin-8 (IL-6, IL-8) mRNA and protein levels of carbon black nanoparticles with concentration gradient after 72 h exposure. Western blot analysis was conducted to detect the expression levels of toll-like receptor 4 (TLR4), phosphorylated nuclear factor-κB (P-NF-κB), apoptosis-related speckled protein (ASC) and Caspase-1 associated with nuclear factor-κB. According to high-throughput sequencing results, differentially expressed Circrnas were screened and identified by qRT-PCR, and those with stable differentially expressed circrnas and the strongest association with the NF-κB pathway were selected for ring performance identification.Results:After being exposed to carbon black nanoparticles for 72 h, the activity of 16HBE cells decreased significantly ( P<0.05), and the release of lactate dehydrogenase increased significantly ( P<0.05). Compared with control group, mRNA expression levels of IL-6 and IL-8, protein levels of IL-6 and IL-8 were increased, and protein levels of TLR4, p-NF-κB, ASC and Caspase-1 were significantly up-regulated in 16 HBE cells of different concentrations, with statistical significance ( P<0.05). Compared with the control group, a total of 492 differentially expressed circular Rnas (|log2 FC|>1) were detected. Among the 5 differentially expressed ( P<0.05) circular Rnas, circ_002642 was selected as the object of subsequent research on circular Rnas, affter 72 hours of exposure to 80 μg/ml CBNPs, 16HBE cells showed signlficantly higher expression of circ_002642 ( P<0.05) . Conclusion:Carbon black nanoparticles can induce differentially expressed circular RNAs associated with inflammatory response in human bronchial epithelial cells.

Objective:To investigate the difference and characteristics of autoantibodies expression in patients infected by 2019-nCoV with various severity, and explore the associations between expression profile of autoantibodies and prognosis of COVID-19 patients.Methods:This retrospective study was conducted on patients with COVID-19 admitted to Zhongnan Hospital, Wuhan University from January 30, 2020 to March 16, 2020. Data on medical records, expression of autoantibodies including antinuclear antibody profile (ANA), anticardiolipin antibody (ACA), inflammatory factor and other laboratory indexes were collected and analyzed. The age and sex matched disease controls (cases of pulmonary infection unrelated to 2019-nCoV infection and autoimmune disease) and healthy controls (healthy check-up individuals) were also included. Following groups were established, ANA test groups: 72 cases of COVID-19 group (including 17 critical and severe cases, and 55 mild cases), 37 disease controls and 44 healthy controls; ACA test groups: 111 cases of COVID-19 group (including 37 critical and severe cases, and 74 mild cases), 37 disease controls and 40 healthy controls. The difference of positive rate or expression level of autoantibodies among various groups was analyzed, and the difference of inflammatory biomarkers and other parameters were compared between patients with ANA positive results and negative results. The Spearman correlation test was applied to determine the relationship between ACA and other parameters. Kaplan-Meier estimation was used to plot survival curves, the log-rank analysis was utilized to explore the association between antibodies and outcome of COVID-19 patients.Results:The positive rate of antibodies was significantly higher in the COVID-19 group than disease and healthy control groups, the ANA fluorescence: 22.22% (16/72), 5.41% (2/37), 6.82% (3/44); ANA spectrum:26.39% (19/72), 8.11% (3/37), 9.09% (4/44); and ACA:37.84% (42/111), 8.11% (3/37), 5.00% (2/40); all P<0.05. The positive rate of ANA, ACA-IgM and the expression level of ACA-IgM were significantly higher in severe COVID-19 subgroups (critically and severe COVID-19 patients) than in the mild COVID-19 patients (the ANA fluorescence: 47.06% [8/17] vs. 14.55% [8/55], ANA spectrum:66.67% [9/17] vs. 18.18% [10/55], ACA-IgM:30.43% [10/37] vs. 9.46% [7/74]; all P<0.05). There were significant differences in the number of red blood cells, hemoglobin concentration, hematocrit, activated partial thromboplastin time, C-reactive protein, interleukin-6 and serum amyloid A between COVID-19 ANA-positive group and COVID-19 ANA-negative group (all P<0.05). The level of ACA-IgM was positively correlated with white blood cell count ( r=0.354, P<0.001), neutrophil count ( r=0.344, P<0.001), platelet count ( r=0.198, P=0.038), D-Dimer ( r=0.260, P=0.009), glutamic-pyruvic transaminase ( r=0.214, P=0.024), γ-glutamyl transpeptidase ( r=0.283, P=0.003), blood urea nitrogen ( r=0.223, P=0.019), and negatively correlated with superoxide dismutase ( r=-0.228, P=0.020). Survival analysis showed that cumulative survival rate of event-free survival (EFS) was lower in patients with positive ANA/ACA-IgM results than in patients with negative ANA/ACA-IgM results ( P<0.05). Conclusions:ANA and ACA autoantibodies can be detected in COVID-19 patients. The positive rate and the expression level of ANA and ACA increase in proportion with the severity of COVID-19 patients. ANA and ACA-IgM could be used as risk stratification determinants for predicting survival of COVID-19 patients.

Since the outbreak of the novel coronavirus pneumonia (COVID-19), it is urgently to develop the effective strategies for its clinical test or treatments world widely. Extracellular vesicles (EV)/exosomes could function as effective carriers for intercellular communication. Increasing evidence revealed that mesenchymal stem cell-derived EV/exosomes could be considered as an alternative cell-free therapy against the acute respiratory distress syndrome. Moreover, the EV-related metabolomics, proteomics, relapsing, deep-vein-thrombosis, myocardial-toxicity, liquid-biopsy and bio-therapeutic researches focusing on the COVID-19 will not only extended our knowledge for the diagnosis, but also provide novel ideas for treatment of this fatal disease. This article will systemically review the progresses of EV/exosomes in the diagnosis and treatment of COVID-19.

OBJECTIVE：To estab lish a method that can comprehensively and rapidly analyze the chemical compositions of Miao medicine Caesalpinia decapetala，and to providing reference for quality control and pharmacodynamic material basis study of C. decapetala . METHODS ：UPLC-Q-TOF-MS/MS was adopted . The determination was performed on Agilent SB-C 18 column with mobile phase consisted of 0.1％ formic acid solution- 0.1％ formic acid acetonitrile （gradient elution ）at the flow rate of 0.25 mL/min. The column temperature was 30 ℃，and sample size was 2 µL. ESI source was applied in negative and positive scanning ion mode and data collection range of m/z 50-1 500. The capillary voltage was 4.5 kV，the atomizing gas （nitrogen）pressure was 1.2 Bar， the solvent removal gas was nitrogen ，the flow rate of solvent removal gas was 8 L/min and the solvent removal gas temperature was 200 ℃. Data Analysis 4.2 software was adopted to analyze fragment ion information of each peak ，and identify chemica l compositions on the basis of relevant literature and mass spectograms of reference substance. RESULTS ：Under positive ion mode ， 9 chemical compounds were identified ；peak 1，2，3，4，5，6，7，8，9 were catechin ，protohematoxylin B ，epicatechin，ethyl gallate，quercetin，luteolin，3-deoxy-hematoxylin chalcone ， isoliquiritigenin and linoleic acid. Under negative ion mode ， U1812403）， totally 21 peaks were confirmed and 13 compounds were identified；peak 3，4，5，6，7，8，9，10，11，12，13，15， 21 were catechins ， brevifolin carboxylic acid ， proto- hematoxylin B ，epicatechin，ethyl gallate ，epicatechin gallate ， quercetin，resveratrol，hematoxylin，luteolin，3-deoxy-hema- toxylin， isoliquiritigenin， linoleic acid. CONCLUSIONS UPLC-Q-TOF-MS/MS method is established successfully for analysis of chemical compositions in C. decapetala .

OBJECTIVE： To study the protective effects of Polygonum orientale extract on myocardial ischemia-reperfusion injury （MIRI） model rats， and to provide reference for it’s deeply development of medicinal source. METHODS： Totally 24 rats were randomly divided into sham operation group （normal saline）， model group （normal saline）， Compound danshen tablet group （positive group， 0.17 g/kg） and P. orientale extract group （86 g/kg， calculated by crude drug）， with 6 rats in each group. All groups were given drugs 2 mL/100 g intragastrically once a day. After 4 d of consecutive administration， MIRI model was induced by the left anterior descending branch of arteria coronaria in all groups except for sham operation group. 24 h after reperfusion， they were given related medicine again. After medication， the changes of electrocardiogram ST segment were monitored in each group. The plasma levels of LDH， CK-MB， CK， cTn-I， SOD and NO were detected in each group. The myocardial infarction rate in each group was calculated and the pathomorphological changes in the myocardium were observed. RESULTS： Compared with sham operation group， ST segment of myocardial electrocardiogram was increased in model group （P＜0.01）. The plasma levels of LDH， CK， CK-MB and cTn-I were increased significantly （P＜0.01）， while the plasma levels of SOD and NO were decreased significantly （P＜0.01）. The rate of myocardial infarction was increased significantly （P＜0.01）， and pathomorphological changes were observed in myocardial tissue such as infiltration of inflammatory cells and loose cytoplasm of cardiac myocytes. Compared with model group， ST segment of myocardial electrocardiogram was decreased significantly in Compound danshen tablet group and P. orientale extract group （P＜0.05）； the plasma levels of LDH， CK， CK-MB and cTn-I were decreased significantly （P＜0.05）， while the plasma levels of SOD and NO were increased significantly （P＜0.05）； the rate of myocardial infarction was decreased significantly （P＜0.05）， and inflammatory cell infiltration and tissue edema in myocardium were relieved to varying degrees. CONCLUSIONS： The protective effect of P. orientale extract protect on MIRI may be exerted by anti-oxidative damage.