OBJECTIVE: Anemoside B4 (AB4), the most abundant triterpenoidal saponin isolated from Pulsatilla chinensis, inhibited influenza virus FM1 or Klebsiella pneumoniae-induced pneumonia. However, the anti-SARS-CoV-2 effect of AB4 has not been unraveled. Therefore, this study aimed to determine the antiviral activity and potential mechanism of AB4 in inhibiting human coronavirus SARS-CoV-2 in vivo and in vitro. METHODS: The cytotoxicity of AB4 was evaluated using the Cell Counting Kit-8 (CCK8) assay. SARS-CoV-2 infected HEK293T, HPAEpiC, and Vero E6 cells were used for in vitro assays. The antiviral effect of AB4 in vivo was evaluated by SARS-CoV-2-infected hACE2-IRES-luc transgenic mouse model. Furthermore, label-free quantitative proteomics and bioinformatic analysis were performed to explore the potential antiviral mechanism of action of AB4. Type I IFN signaling-associated proteins were assessed using Western blotting or immumohistochemical staining. RESULTS: The data showed that AB4 reduced the propagation of SARS-CoV-2 along with the decreased Nucleocapsid protein (N), Spike protein (S), and 3C-like protease (3CLpro) in HEK293T cells. In vivo antiviral activity data revealed that AB4 inhibited viral replication and relieved pneumonia in a SARS-CoV-2 infected mouse model. We further disclosed that the antiviral activity of AB4 was associated with the enhanced interferon (IFN)-β response via the activation of retinoic acid-inducible gene I (RIG-1) like receptor (RLP) pathways. Additionally, label-free quantitative proteomic analyses discovered that 17 proteins were significantly altered by AB4 in the SARS-CoV-2 coronavirus infections cells. These proteins mainly clustered in RNA metabolism. CONCLUSION Our results indicated that AB4 inhibited SARS-CoV-2 replication through the RLR pathways and moderated the RNA metabolism, suggesting that it would be a potential lead compound for the development of anti-SARS-CoV-2 drugs.

Objective: To investigate the prevalence and associated factors of depressive symptoms among middle and high school students in Zhejiang Province, so as to provide scientific basis for the implementation of depressive intervention. Methods: Based on the health status and associated factors of middle and high school students in the project "Monitoring of Common Diseases and Health Influencing Factors of Students" during 2018 to 2021, a total of 73 309 students including middle school, ordinary high school and vocational high school surveyed in 11 cities of Zhejiang Province were selected by multi stage stratified cluster random sampling method. From 2018 to 2021, there were 6 008, 21 917, 23 712 and 21 672 students, respectively. The Chi square test and Logistic regression model were used to analyze the influencing factors of depressive symptoms in middle and high school students. Results: From 2018 to 2021, depressive symptoms detection rate of middle school students was 14.8%, with higher rate in girls (17.1%) than in boys (12.7%), higher rate in high school (17.1% in ordinary high school, 17.6% in vocational high school) than middle school (12.5%)( χ 2=278.77, 327.22, P <0.05). Univariate analysis showed that there were statistically significant differences in depressive symptoms detection rate among middle school students with different years (2018: 16.7%，2019: 17.9% , 2020: 13.1%, 2021: 13.0%), residence (yes: 16.3%, no:13.5%), body mass index classification (not overweight or obesity: 14.8%, overweight: 14.2%, Obesity: 15.7%), weekly exercise days (0-2 d: 17.1%, 3-5 d: 12.5%, 6-7 d: 13.1%) and bullying (yes: 35.5%, no: 10.7%) ( χ 2=293.40, 118.35, 7.83, 287.24, 4 978.84, P <0.05). Multivariate Logistic regression analysis showed that female students, ordinary high schools, vocational high schools, obesity, school bullying were positively correlated with depression ( OR =1.65, 1.70, 1.60, 1.12, 5.21), exercise 3 to 5 days per week, exercise 6 to 7 days per week were negatively correlated with depression ( OR=0.77, 0.81, P <0.01). Conclusions Depressive symptoms among middle and high school students in Zhejiang Province are prominent. Strengthening mental health education for students and providing attention and support from families, schools, and society are essential steps to reduce the occurrence of depressive symptoms among these students.

Objective: To describe the prevalence characteristics and trend of malnutrition among primary and secondary school students in Zhejiang Province from 2008 to 2021, so as to provide scientific references for targeted interventions on malnutrition among children and adolescents. Methods: Based on the National Student Common Diseases and Health Influencing Factors Surveillance Project, 81 228 primary and middle school students aged 9-17 from Zhejiang Province were recruited for a questionnaire in 2008, 2014 and 2021, with stratified cluster random sampling method. Malnutrition was determined by Screening for Malnutrition in School aged Children and Adolescents. The Kruskal-Wallis test was used for non normally distributed data, and the Chi square test was used for categorical data. A trend Chi square test analyzed detection rates across different years. Results: The prevalence rates of malnutrition, stunting, mild wasting, and moderate to severe wasting among primary and secondary school students in 2008, 2014 and 2021 were 12.0%, 6.6%, 6.0%; 1.2%, 0.5%, 0.3%; 6.0%, 3.9%, 3.6%; and 4.7%, 2.2%, 2.2%, with an overall declining trend ( χ 2 trend =532.73, 181.43, 161.24, 240.38, P <0.05). The prevalence rates of malnutrition and mild wasting in each year were higher in boys (13.7%, 7.6%; 7.5%, 5.0%; 7.1%, 4.5%) than in girls (10.3%, 4.4%; 5.5%, 2.7%; 4.8%, 2.5%) ( χ 2=54.45, 88.67; 47.04, 81.07; 85.28, 98.81; P <0.01). The difference in malnutrition prevalence between urban and rural areas gradually narrowed (12.5%, 11.6%; 6.9%, 6.3%; 6.0%, 6.0%), with no statistically significant difference in 2021 ( χ 2= 0.01 , P >0.05). Malnutrition among primary and secondary school students was primarily characterized by mild wasting (56.0%) in Zhejiang Province. Compared to 2008, the prevalence of malnutrition in 2014 and 2021 showed a steady upward trend with increasing age in Zhejiang Province( χ 2 trends =44.52, 11.78, P <0.01). Conclusions The prevalence of malnutrition among primary and secondary school students aged 9 to 17 years in Zhejiang Province decreases by year from 2008 to 2021. However, the prevalence increase steadily with age, and boys have a higher prevalence of malnutrition. Policies should be developed age , gender , and growth appropriate dietary patterns to reduce malnutrition among primary and secondary school students.

Objective:To investigate the causality between intestinal flora and hypertrophic scars (HS) of human.Methods:This study was a study based on two-sample Mendelian randomization (TSMR) analysis. The data on intestinal flora ( n=18 473) and HS ( n=208 248) of human were obtained from the genome-wide association study database. Genetically variable genes at five levels (phylum, class, order, family, and genus) of known intestinal flora, i.e., single nucleotide polymorphisms (SNPs), were extracted as instrumental variables for linkage disequilibrium (LD) analysis. Human genotype-phenotype association analysis was performed using PhenoScanner V2 database to exclude SNPs unrelated to HS in intestinal flora and analyze whether the selected SNPs were weak instrumental variables. The causal relationship between intestinal flora SNPs and HS was analyzed through four methods of TSMR analysis, namely inverse variance weighted (IVW), MR-Egger regression, weighted median, and weighted mode. Scatter plots of significant results from the four aforementioned analysis methods were plotted to analyze the correlation between intestinal flora SNPs and HS. Both IVW test and MR-Egger regression test were used to assess the heterogeneity of intestinal flora SNPs, MR-Egger regression test and MR-PRESSO outlier test were used to assess the horizontal multiplicity of intestinal flora SNPs, and leave-one-out sensitivity analysis was used to determine whether HS was caused by a single SNP in the intestinal flora. Reverse TSMR analyses were performed for HS SNPs and genus Intestinimonas or genus Ruminococcus2, respectively, to detect whether there was reverse causality between them. Results:A total of 196 known intestinal flora, belonging to 9 phyla, 16 classes, 20 orders, 32 families, and 119 genera, were obtained, and multiple SNPs were obtained from each flora as instrumental variables. LD analysis showed that the SNPs of the intestinal flora were consistent with the hypothesis that genetic variation was strongly associated with exposure factors, except for rs1000888, rs12566247, and rs994794. Human genotype-phenotype association analysis showed that none of the selected SNPs after LD analysis was excluded and there were no weak instrumental variables. IVW, MR-Egger regression, weighted median, and weighted mode of TSMR analysis showed that both genus Intestinimonas and genus Ruminococcus2 were causally associated with HS. Among them, forest plots of IVW and MR-Egger regression analyses also showed that 16 SNPs (the same SNPs number of this genus below) of genus Intestinimonas and 15 SNPs (the same SNPs number of this genus below) of genus Ruminococcus2 were protective factors for HS. Further, IVW analysis showed that genus Intestinimonas SNPs (with odds ratio of 0.62, 95% confidence interval of 0.41-0.93, P<0.05) and genus Ruminococcus2 SNPs (with odds ratio of 0.62, 95% confidence interval of 0.40-0.97, P<0.05) were negatively correlated with the risk of HS. Scatter plots showed that SNPs of genus Intestinimonas and genus Ruminococcus2 were protective factors of HS. Both IVW test and MR-Egger regression test showed that SNPs of genus Intestinimonas (with Q values of 5.73 and 5.76, respectively, P>0.05) and genus Ruminococcus2 (with Q values of 13.67 and 15.61, respectively, P>0.05) were not heterogeneous. MR-Egger regression test showed that the SNPs of genus Intestinimonas and genus Ruminococcus2 had no horizontal multiplicity (with intercepts of 0.01 and 0.06, respectively, P>0.05); MR-PRESSO outlier test showed that the SNPs of genus Intestinimonas and genus Ruminococcus2 had no horizontal multiplicity ( P>0.05). Leave-one-out sensitivity analysis showed that no single intestinal flora SNP drove the occurrence of HS. Reverse TSMR analysis showed no reverse causality between HS SNPs and genus Intestinimonas or genus Ruminococcus2 (with odds ratios of 1.01 and 0.99, respectively, 95% confidence intervals of 0.97-1.06 and 0.96-1.04, respectively, P>0.05). Conclusions:There is a causal relationship between intestinal flora and HS of human, in which genus Intestinimonas and genus Ruminococcus2 have a certain effect on inhibiting HS.

ObjectiveTo establish the identification method of Dalbergiae Odoriferae Lignum（DOL） and its counterfeits by nuclear magnetic resonance hydrogen spectrum（¹H-NMR） combined with multivariate statistical analysis. Method¹H-NMR spectra of DOL and its counterfeits were obtained by NMR， and the full composition information was established and transformed into a data matrix， and the detection conditions were as follows：taking dimethyl sulfoxide-d₆（DMSO-d₆） containing 0.03% tetramethylsilane（TMS） as the solvent， the constant temperature at 298 K（1 K=-272.15 ℃）， pulse interval of 1.00 s， spectrum width of 12 019.23 Hz， the scanning number of 16 times， and the sampling time of 1.08 s. Similarity examination and hierarchical cluster analysis（HCA） were performed on the data matrix of DOL and its counterfeits， and orthogonal partial least squares-discriminant analysis（OPLS-DA） was used to analyze the data matrix and identify the differential components between them. In the established OPLS-DA category variable value model， the category variable value of DOL was set as 1， and the category variable value of the counterfeits was set as 0， and the threshold was set as ±0.3， in order to identify the commercially available DOL. The OPLS-DA score plot was used to determine the types of counterfeits in commercially available DOL， and it was verified by thin layer chromatography（TLC）. ResultThe results of similarity analysis and HCA showed that there was a significant difference between DOL and its counterfeits. OPLS-DA found that the differential component between DOL and its counterfeits was trans-nerolidol. The established category variable value model could successfully identify the authenticity of the commercially available DOL. The results of the OPLS-DA score plot showed that there were heartwood of Dalbergia pinnata and D. cochinchinensis in the commercially available DOL， and were consistent with the TLC verification results. ConclusionThere is a phenomenon that heartwood of D. pinnata and D. cochinchinensis are sold as DOL in the market. ¹H-NMR combined with multivariate statistical analysis can effectively distinguish DOL and its counterfeits， which can provide a reference for the identification of them.

Background::The calcineurin inhibitor (CNI)-based immune maintenance regimen that is commonly used after renal transplantation has greatly improved early graft survival after transplantation; however, the long-term prognosis of grafts has not been significantly improved. The nephrotoxicity of CNI drugs is one of the main risk factors for the poor long-term prognosis of grafts. Sirolimus (SRL) has been employed as an immunosuppressant in clinical practice for over 20 years and has been found to have no nephrotoxic effects on grafts. Presently, the regimen and timing of SRL application after renal transplantation vary, and clinical data are scarce. Multicenter prospective randomized controlled studies are particularly rare. This study aims to investigate the effects of early conversion to a low-dose CNI combined with SRL on the long-term prognosis of renal transplantation.Methods::Patients who receive four weeks of a standard regimen with CNI + mycophenolic acid (MPA) + glucocorticoid after renal transplantation in multiple transplant centers across China will be included in this study. At week 5, after the operation, patients in the experimental group will receive an additional administration of SRL, a reduction in the CNI drug doses, withdrawal of MPA medication, and maintenance of glucocorticoids. In addition, patients in the control group will receive the maintained standard of care. The patients’ vital signs, routine blood tests, routine urine tests, blood biochemistry, serum creatinine, BK virus (BKV)/cytomegalovirus (CMV), and trough concentrations of CNI drugs and SRL at the baseline and weeks 12, 24, 36, 48, 72, and 104 after conversion will be recorded. Patient survival, graft survival, and estimated glomerular filtration rate will be calculated, and concomitant medications and adverse events will also be recorded.Conclusion::The study data will be utilized to evaluate the efficacy and safety of early conversion to low-dose CNIs combined with SRL in renal transplant patients.Trial registration::Chinese Clinical Trial Registry, ChiCTR1800017277.

Coronavirus is an important pathogen of humans and animals. Among them, the novel coronavirus disease (COVID-19) breaking out in 2019 has brought a fatal threat to human health. The host"s innate immune response is the host"s first line of defense against pathogen invasion, but an excessive immune response can also aggravate viral infection and pathological damage. The immune escape of coronavirus is a critical pathogenic mechanism causing death. This work mainly reviews the pathogenic mechanism of coronavirus immune escape from several aspects such as host immunosensor, interferon, cytokine and coronavirus antagonizing host immune response, which provide a theoretical reference for the development of anti-coronavirus drugs.

Objective:To explore the effects of platelet-rich fibrin (PRF) on the periodontal soft tissue and bone tissue during the treatment of periodontitis.Methods:Twenty patients with three-wall bone defect of periodontitis requiring periodontal surgery in the Department of Stomatology of the Tianjin First Central Hospital from December 2018 to August 2019 were selected and randomly divided into the experimental group ( n=10) and the control group ( n=10) using the random number table method. For the experimental group, the PRF membrane was covered with Bio-Oss bone. For the control group, only Bio-Oss bone powder was implanted. The image measurement values of probing depth (PD), periodontal attachment level (AL), bleeding index (BI) and bone defect (IBD) were recorded before and 6 months after the operation, and statistical analysis was performed. Results:The measured values of PD, AL and IBD of the two groups after the operation were better than those before the operation (all P<0.05), which indicated that the operation was effective. At 6 months after the operation, the PD and AL of the experimental group were better than those of the control group (all P<0.05). However, BI and IBD had no statistical significance between the two groups (all P>0.05). Conclusions:The combined use of PRF membrane during periodontal bone grafting can effectively reduce PD and obtain more new clinical attachments.

A series of 2-(((5-akly/aryl-1-pyrazol-3-yl)methyl)thio)-5-alkyl-6-(cyclohexylmethyl)-pyrimidin-4(3)-ones were synthesized and their anti-HIV-1 activities were evaluated. Most of these compounds were highly active against wild-type (WT) HIV-1 strain (IIIB) with EC values in the range of 0.0038-0.4759 μmol/L. Among those compounds, had an EC value of 3.8 nmol/L and SI (selectivity index) of up to 25,468 indicating excellent activity against WT HIV-1. anti-HIV-1 activity and resistance profile studies suggested that compounds and displayed potential anti-HIV-1 activity against laboratory adapted strains and primary isolated strains including different subtypes and tropism strains (ECs range from 4.3 to 63.6 nmol/L and 18.9-219.3 nmol/L, respectively). On the other hand, it was observed that those two compounds were less effective with EC values of 2.77 and 4.87 μmol/L for HIV-1A (K103N + Y181C). The activity against reverse transcriptase (RT) was also evaluated for those compounds. Both and obtained sub-micromolar IC values showing their potential in RT inhibition. The pharmacokinetics examination in rats indicated that compound has acceptable pharmacokinetic properties and bioavailability. Preliminary structure-activity relationships and molecular modeling studies were also discussed.

OBJECTIVE： To establish a quantitative analysis of multi-components by singer marker （QAMS） for determining the contents of diosgenin， 5-hydroxymethylfurfural， vanillic acid， rutin， quercetin， kaempferol in Polygonati Rhizoma and its decoction piece. METHODS： HPLC external standard method was used to determine the contents of 6 components in Polygonati Rhizoma and its decoction piece simultaneously [the separation was carried out on Diamonsil-C18 column with mobile phase consisted of acetonitrile-water （gradient elution）； the detection wavelengths of 5-hydroxymethylfurfural， vanillic acid， rutin， quercetin and kaempferol were set at 254 nm（0-60 min）； the detection wavelength of diosgenin was set at 202 nm （60-75 min） at the flow rate of 1.0 mL/min. The column temperature was 30 ℃]. Using vanillic acid as internal standard， relative correction factors （RCFs） of aother 5 components were calculated and to investigate durability. Relative retention method was used to accurately locate the chromatographic peaks of the components to be determined， and then the contents of the aother 5 components in Polygonati Rhizoma were calculated according to RCFs， and the results were compared with those determined by external standard method. The method was validated by Polygonati Rhizoma decoction piece. The contents of 6 components were determined by QAMS method and external standard method respectively， and then the differences of content determination were compared between 2 methods. RESULTS： The methodology investigation results of HPLC method were in line with related requirements. Within the linear range， the RCFs of diosgenin， 5-hydroxymethylfurfural， rutin， quercetin， kaempferol were 0.195， 0.025， 0.263， 0.345 and 0.075， respectively. Under different experiment conditions， RCFs showed good reproducibility； there was no statistical significance of 6 components in Polygonati Rhizoma and its decoction piece determined by external standard method and QAMS method （P＞0.05）. CONCLUSIONS： Established QAMS method is suitable for simultaneous determination of 6 components in Polygonati Rhizoma and its decoction piece.