Objective: To evaluate the long-term healing rate of transsphincteric anal fistula treated with anal fistula plug procedure and the risk factors affecting the healing of anal fistula. Methods: A retrospective case-control study was conducted to analyze the clinical data of 207 patients with transsphincteric anal fistulas who received anal fistula plug procedure at the Department of General Surgery, Beijing Chaoyang Hospital of Capital Medical University from August 2008 to September 2012. Inclusion criteria: (1) consistent with the diagnosis of transsphincteric anal fistula: the anal fistula passed through the internal and external sphincter; (2) complete data; (3) initial treatment with anal fistula plug procedure. Exclusion criteria: (1) acute rectal or perianal infection or poorly controlled focal infection; (2) recent incision and drainage of perianal abscess or spontaneous rupture of abscess; (3) patients with malignant tumor; (4) patients with Crohn′s disease or ulcerative colitis; (5) patients with heart, liver, brain, lung or renal insufficiency; (6) cachexia due to various chronic wasting diseases; (7) patients could not tolerate surgery. Patients were followed up for anal fistula healing. The cumulative healing rate of patients with transsphincteric anal fistula was plotted using the Kaplan-Meier method, and the factors affecting anal fistula healing were explored by univariate and multivariate logistic regression analysis. Results: There were 186 males and 21 females with age of 15 to 69 (mean 38) years. The duration of anal fistula was 3-60 (mean 15) months. Three patients had a history of previous episodes of perianal abscess and underwent incision and drainage of perianal abscess (all more than 3 months). During follow-up ending on October 31, 2018, 72 patients (34.8%) were lost to follow-up. Among 135 patients who were successfully followed up, the average follow-up period was 96 (75-124) months. Seventy-five patients had anal fistula healing, with healing rate of 55.6%. Kaplan-Meier survival curve showed that the healing time of anal fistula was prolonged and finally stabilized at 55.6%. In the patients who failed initial treatment with anal fistula plug packing, there were 6 cases whose anal fistula healed spontaneously without other treatment. Among them, 3 cases healed spontaneously 2 years and 3 cases 3 years after operation without recurrence. From 2008 to 2012, the annual healing rates of anal fistula plug treatment were 3/6, 61.5% (24/39), 42.1% (24/57), 12/15 and 12/18, respectively. Multivariate logistic regression analysis showed that the duration of anal fistula≥6 months (OR=3.187, 95% CI: 1.361-7.466, P=0.008) was an independent risk factor for anal fistula healing after treatment with anal fistula plug. Conclusion The long-term efficacy of anal fistula plug procedure in the treatment of transsphincteric anal fistula is positive, and this procedure should be implemented as soon as possible.

Objective:To evaluate the long-term effects of anal fistula plug treatment on postoperative anal function in patients with trans-sphincteric perianal fistula, and identify risk factors associated with anal function.Methods:A case-control study was conducted. Clinical and follow-up data of 123 patients with trans-sphincteric perianal fistula receiving anal fistula plug treatment in Beijing Chaoyang Hospital from August 2008 to September 2012 were retrospectively analyzed. The follow-up deadline was April 30, 2020. The Wexner score for incontinence was used to evaluate pre-and postoperative anal function (range from 0 to 20, with higher score representing worse function). The potential risk factors affecting postoperative anal function, including gender, age, fasting blood glucose, diabetes, smoking, alcoholism, location of external opening of anal fistula, surgeon expertise and operation time, were statistically analyzed.Results:Among the 123 patients, 114 were male and 9 were female, the median age was 39 (15-69) years, body mass index (BMI) was (26.2±3.9) kg/m 2, and the median distance between the external opening of anal fistula and the anal verge was 2.6 (1.0-5.0) cm. The median operation time was 30.4 (15.0-60.0) minutes. The median follow-up time was 120 (93-141) months. Sixty-nine patients (56.5%) were healed. The assessment of anal function by Wexner incontinence score showed that 33 (26.8%) patients had anal functional decline after surgery, and the postoperative median Wexner score was 1.34 (0-8), which was significantly higher than preoperative score of 0.17 (0-4) ( Z=-5.057, P<0.001). Compared with preoperative levers, postoperative subscores of flatus incontinence, liquid incontinence, solid incontinence and alteration in lifestyle were increased significantly (all P<0.05). Subgroup analysis showed that both in the healed and unhealed groups, the postoperative Wexner scores were higher than those before surgery [healed group: 1.22 (0-8) vs. 0.17 (0-1), Z=-3.796, P<0.001; unhealed group: 1.5 (0-8) vs.0.17 (0-3), Z=-3.422, P=0.001]. Univariate analysis revealed that 33 patients with postoperative anal functional decline had higher BMI, higher fasting blood glucose, higher proportion of alcoholism, longer distance between external opening of anal fistula and the anal verge, and longer operation time (all P<0.05). Multivariate logistic regression analysis testified that higher BMI (OR=1.485, 95% CI: 1.220-1.807, P<0.001) and longer distance between external opening and anal verge (OR=2.207, 95% CI: 1.276-3.220, P=0.003) were independent risk factors for postoperative anal functional decline. Conclusions:The treatment for trans-sphincteric anal fistula with anal fistula plug leads to long-term postoperative anal function decline. For patients with obesity and longer distance between external opening of anal fistula and the anal verge, this procedure should be performed with particular caution, and the anal sphincter should be preserved as much as possible during the operation.

Objective:To evaluate the long-term effects of anal fistula plug treatment on postoperative anal function in patients with trans-sphincteric perianal fistula, and identify risk factors associated with anal function.Methods:A case-control study was conducted. Clinical and follow-up data of 123 patients with trans-sphincteric perianal fistula receiving anal fistula plug treatment in Beijing Chaoyang Hospital from August 2008 to September 2012 were retrospectively analyzed. The follow-up deadline was April 30, 2020. The Wexner score for incontinence was used to evaluate pre-and postoperative anal function (range from 0 to 20, with higher score representing worse function). The potential risk factors affecting postoperative anal function, including gender, age, fasting blood glucose, diabetes, smoking, alcoholism, location of external opening of anal fistula, surgeon expertise and operation time, were statistically analyzed.Results:Among the 123 patients, 114 were male and 9 were female, the median age was 39 (15-69) years, body mass index (BMI) was (26.2±3.9) kg/m 2, and the median distance between the external opening of anal fistula and the anal verge was 2.6 (1.0-5.0) cm. The median operation time was 30.4 (15.0-60.0) minutes. The median follow-up time was 120 (93-141) months. Sixty-nine patients (56.5%) were healed. The assessment of anal function by Wexner incontinence score showed that 33 (26.8%) patients had anal functional decline after surgery, and the postoperative median Wexner score was 1.34 (0-8), which was significantly higher than preoperative score of 0.17 (0-4) ( Z=-5.057, P<0.001). Compared with preoperative levers, postoperative subscores of flatus incontinence, liquid incontinence, solid incontinence and alteration in lifestyle were increased significantly (all P<0.05). Subgroup analysis showed that both in the healed and unhealed groups, the postoperative Wexner scores were higher than those before surgery [healed group: 1.22 (0-8) vs. 0.17 (0-1), Z=-3.796, P<0.001; unhealed group: 1.5 (0-8) vs.0.17 (0-3), Z=-3.422, P=0.001]. Univariate analysis revealed that 33 patients with postoperative anal functional decline had higher BMI, higher fasting blood glucose, higher proportion of alcoholism, longer distance between external opening of anal fistula and the anal verge, and longer operation time (all P<0.05). Multivariate logistic regression analysis testified that higher BMI (OR=1.485, 95% CI: 1.220-1.807, P<0.001) and longer distance between external opening and anal verge (OR=2.207, 95% CI: 1.276-3.220, P=0.003) were independent risk factors for postoperative anal functional decline. Conclusions:The treatment for trans-sphincteric anal fistula with anal fistula plug leads to long-term postoperative anal function decline. For patients with obesity and longer distance between external opening of anal fistula and the anal verge, this procedure should be performed with particular caution, and the anal sphincter should be preserved as much as possible during the operation.

Objective:To evaluate the immunogenicity and protective effect of a multicomponent recombinant protein vaccine EPRHP014 constructed independently and provide a scientific basis for developing new tuberculosis (TB) vaccine and effective prevention and control of TB.Methods:Three full-length Mycobacterium ( M.) tuberculosis protein antigens (EsxH, Rv2628, and HspX) and two epitope-predicted and optimized epitope-dominant protein antigens (nPPE18 and nPstS1) were selected, from which five protein antigens were used to construct a protein antigen composition EPRHP014, including a fusion expression multi-component protein antigen (EPRHP014f) and a multi-component mixed protein antigen (EPRHP014m) formed with the five single protein using clone, purification, and purification respectively. Multicomponent protein vaccines EPRHP014f and EPRHP014m were prepared with aluminum adjuvant, and the BCG vaccine was used as a control. ELISA detected the titer of serum-specific antibodies, the secretion of various cytokines was detected by ELISpot and Luminex, and immune protection was observed by the M.tuberculosis growth inhibition test in vitro. The results were statistically analyzed by t-test or rank sum test, and P<0.05 was considered a statistically significant difference. Results:Mice Immunized with EPRHP014m and EPRHP014f could produce highly effective IgG antibodies and their subtypes IgG1 and IgG2a, and the antibody titers were similar to those of mice immunized with BCG, with no statistical significance ( P>0.05). The number of spot-forming cells (SFC) secreting IFN-γ and IL-4 induced by EPRHP014f group was significantly higher than those by EPRHP014m group and BCG group ( P<0.05), but there was no significant difference in the number of SFC for IFN-γ and IL-4 induced between EPRHP014m group and BCG group ( P>0.05). The secretion levels of GM-CSF and IL-12p70 induced by the EPRHP014m group were higher than those of the BCG group ( P<0.05), but there was no significant difference in the levels of IL-6 and IL-10 induced between EPRHP014m group and BCG group ( P>0.05). There was no significant difference in the secretions of IL-6, IL-10, IL-12, and GM-CSF between the EPRHP014f and BCG groups ( P>0.05). EPRHP014m group, EPRHP014f group, and BCG group had obvious antibacterial effects in vitro, and the difference was insignificant ( P>0.05). Conclusion:Both EPRHP014f and EPRHP014m can induce strong humoral and cellular immune responses in mice after immunization, and have a strong ability to inhibit the growth of M. tuberculosis in vitro, indicating that the antigen composition EPRHP014 has good potential in the development and application of TB vaccine.

Objective:To preliminarily evaluate the immunogenicity and efficacy of two novel tuberculosis vaccine candidates (a fusion multicomponent protein EPDPA015f and a mixed multicomponent protein EPDPA015m) and to provide a new antigen combination for the development of tuberculosis vaccines.Methods:Recombinant plasmids for the expression of EPDPA015f and EPDPA015m proteins were constructed. Six-week-old BALB/c mice were immunized with EPDPA015f or EPDPA015m in combination with aluminium adjuvant (50 μg/mouse) for three times with an interval of 10 d. The mice were sacrificed 10 d after the last immunization to collect blood and spleen samples. Serum antibody titers and cytokine levels were measured by ELISA, Luminex technique and enzyme-linked immunospot assay (ELISPOT). Mycobacterial growth inhibition assay (MGIA) was used to detect the ability of mouse splenocytes to inhibit the growth of Mtb in vitro. One-way analysis of variance and t-test were used for statistical analysis. Results:Both EPDPA015f and EPDPA015m could induce the production of various cytokines and IgG antibodies at a high level. The levels of cytokines related to Th1 (IL-2, TNF-α, IFN-γ), Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) as well as other proinflammatory cytokines (GM-CSF, IL-12) were higher in the EPDPA015f group than in the adjuvant group ( P<0.05). The titer of IgG antibody induced by EPDPA015f was as high as 1∶4×10 6. The results of MGIA showed that the numbers of Mtb (lgCFU) in the PBS, adjuvant, EPDPA015f and EPDPA015m groups were 3.46±0.11, 3.51±0.06, 2.98±0.09 and 3.19±0.08, respectively. The number of colonies in the EPDPA015f group was the least as compared with that in the other three groups ( P<0.001, P<0.001, P<0.01). Conclusions:The vaccine candidate EPDPA015f could elicit more comprehensive and high-level cellular and humoral immune responses, and exhibited superior in vitro inhibitory activity against the growth of Mtb. EPDPA015f had the potential to be used as a preventive vaccine or a booster vaccine

OBJECTIVE To analyze the serum chemical composition of rats after intragastric administration of water extract of crude Anemarrhenae Rhizoma-crude Phellodendri Chinensis and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis based on liquid chromatography-mass spectrometry(LC-MS)technology,predict the effect of salt processing on the treatment of type 2 dia-betes in Anemarrhenae Rhizoma-Pheellodendri Chinensis combined with network pharmacology,and preliminarily verify it through in vitro experiments.METHODS Rats were continuously intragastrically administered with crude Anemarrhenae Rhizoma-crude Phello-dendri Chinensis drug pair and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis drug pair water extract twice,with an in-terval of 1 h.After 60 min of the last administration,the blood was taken from the abdominal aorta,and the protein was precipitated by methanol.After dissolution,the chromatographic column was Shim-pack GIST C18(4.6 mm×150 mm,5 μm);the mobile phase A was 0.1%formic acid water,and the mobile phase B was 0.1%formic acid-acetonitrile;gradient elution,positive and negative ion full scan mode,mass scan range 100-1 500 m/z.Combined with the secondary spectrum of the database and literature,the blood compo-nents of crude Anemarrhenae Rhizoma-crude Phellodendri Chinensis drug pair and salted Anemarrhenae Rhizoma-salted Phellodendri Chinensis drug pair were analyzed and identified.The disease targets of type 2 diabetes were retrieved,and the protein interaction net-work analysis,GO and KEGG pathway enrichment analysis were performed on the intersection targets of blood components and diseases.The"blood components-targets"network diagram was constructed,and the selected core components and core targets were verified by molecular docking using AutoDock software.In the verification experiment,HepG2 cells were used as the experimental ob-ject,and the insulin resistance model was induced by high insulin and high glucose.CCK8 method was used to test the effect of Rhizo-ma Anemarrhenae-Phellodendri Chinensis on cell proliferation before and after salt processing.Western blot was used to detect the ex-pression of PI3K-AKT signaling pathway-related proteins.RESULTS 15 prototype components and 1 mangiferin metabolic compo-nent were identified in the serum of rats.17 prototype components and 1 mangiferin metabolite were identified in the rat serum of the water extract of Anemarrhenae Rhizoma-Phellodendri Chinensis after salt processing.The contents of mangiferin,berberine and 3-isobutylglutaric acid in the blood components after salt-processing were higher than those in the raw products.According to the results of KEGG and GO,the treatment of type 2 diabetes may be related to the transcriptional regulation of RNA polymerase,inflammatory re-sponse,AGE-RAGE,PI3K-AKT pathway and insulin resistance.Cell experiments showed that the ratio of p-PI3K/PI3K,p-AKT/AKT and GLUT4 protein expression could be up-regulated before and after salt processing,and the effect of salt processing group was better than that of the crude group.CONCLUSION This experiment preliminarily revealed the components of Anemarrhenae Rhizo-ma-Phellodendri Chinensis drug pair entering the blood before and after salt exposure,and suggested that ferulic acid,berberine,ber-berrubine,mangiferin,mTOR,SIRT1,EGFR and PPARA may be the main components and targets of Rhizoma Anemarrhenae-Phel-lodendri Chinensis after salt processing to enhance the therapeutic effect of type 2 diabetes.The mechanism may be to enhance the role of PI3K-AKT and other related signaling pathways,providing an important reference for the pharmacodynamic material basis and clini-cal application of Anemarrhenae Rhizoma-Phellodendri Chinensis before and after salt processing.

ObjectiveTo investigate the key compounds affecting the irritation and to clarify the effect of heating and the addition of ginger juice as the auxiliary material during the processing on the irritation of Magnoliae Officinalis Cortex（MOC） by comparing the irritation and composition of volatile oil in MOC and its different processed products. MethodVolatile oil in raw products， water-processed products， ginger-dried products， ginger-fried products（the amounts of ginger were 10%， 50%， respectively） of MOC were extracted by steam distillation and subjected to rabbit eye irritation experiment， and the volatile components of each sample were detected by gas chromatography-mass spectrometry（GC-MS）. Principal component analysis（PCA）and orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to analyze the data of each sample by SIMCA 14.1. The relative contents of different processed products were compared two by two with those of and raw products or ginger-fried products， and the markers that might be related to the irritation were sorted out according to the principles of variable importance in the projection（VIP） value >1 and P<0.05， and the factors influencing the differences in irritation were analyzed. ResultCompared with the blank group， the administration groups all had irritation to the eyes of rabbits， and the degree of irritation was in the order of raw products>water-processed products>ginger-dried products>ginger-fried products（10%）>ginger-fried products（50%）. The results of PCA and OPLS-DA showed that there were differences in the volatile oil from raw products and different processed products. According to VIP value>1 and P<0.05， and combined with the results of eye irritation experiment， ten volatile compounds related to irritation changes were screened out. Among them， cis-cinnamaldehyde was only detected in raw products， the relative contents of β-caryophyllene， （+）-delta-cadinene， α-humulene， γ-muurolene， （-）-isoledene and citral all increased to different degrees， the contents of p-cymene， 1（10）-4-cadinadien-15-ol and β-eudesmol all decreased to different degrees. ConclusionThe irritation of MOC is reduced after heating and processing with ginger juice， and the synergistic effect of both is more effective for reducing irritation. Among the differential markers associated with changes in irritation， the increase in the relative content of citral is closely related to the addition of ginger juice， while the decrease in the relative contents of cis-cinnamaldehyde， p-cymene， 1（10）-4-cadinadien-15-ol is related to heating， and the changes of other components may be related to the synergistic effect of heating and ginger juice.

Nonpharmaceutical interventions (NPIs) have been commonly deployed to prevent and control the spread of the coronavirus disease 2019 (COVID-19), resulting in a worldwide decline in influenza prevalence. However, the influenza risk in China warrants cautious assessment. We conducted a cross-sectional, seroepidemiological study in Shandong Province, Northern China in mid-2021. Hemagglutination inhibition was performed to test antibodies against four influenza vaccine strains. A combination of descriptive and meta-analyses was adopted to compare the seroprevalence of influenza antibodies before and during the COVID-19 pandemic. The overall seroprevalence values against A/H1N1pdm09, A/H3N2, B/Victoria, and B/Yamagata were 17.8% (95% CI 16.2%-19.5%), 23.5% (95% CI 21.7%-25.4%), 7.6% (95% CI 6.6%-8.7%), and 15.0 (95% CI 13.5%-16.5%), respectively, in the study period. The overall vaccination rate was extremely low (2.6%). Our results revealed that antibody titers in vaccinated participants were significantly higher than those in unvaccinated individuals (P < 0.001). Notably, the meta-analysis showed that antibodies against A/H1N1pdm09 and A/H3N2 were significantly low in adults after the COVID-19 pandemic (P < 0.01). Increasing vaccination rates and maintaining NPIs are recommended to prevent an elevated influenza risk in China.