The objective of this study was to optimise the extraction process of peptide of Poecilobdella manillensis by the Box-Behnken design-response surface methodology, and to investigate its whitening and anti-aging effects. Based on single factor experiments, NaCl solution concentration, extracting time and extracting times were taken as influencing factors, and peptide yield was used as the response value. The response surface model was designed and used to obtain the optimal extraction conditions of peptides of Poecilobdella manillensis: NaCl solution concentration of 4.3%, ultrasonic time of 4 h, and ultrasonic times of 2 times (2 + 2 h), which was significant and well-fitted to the actual experiment. The tyrosinase inhibition activity of peptide of Poecilobdella manillensis was evaluated by measuring the oxidation rate of levodopa (L-DOPA) catalyzed by tyrosinase. Moreover, using Caenorhabditis elegans as a model organism, the effects of peptide of Poecilobdella manillensis on body length, locomotion, reproductive capacity, reactive oxygen species (ROS), and lipofuscin levels were determined. The results showed that peptide of Poecilobdella manillensis exhibited a significant inhibitory effect on tyrosinase, with an IC₅₀ of 0.58 mg·mL^-1, which was stronger than that of the positive control arbutin (2.24 mg·mL^-1). Compared to the control group, 0.1, 0.5 and 1.0 mg·mL^-1 peptide of Poecilobdella manillensis showed no significant differences in the body length, eggs, and body bending of Caenorhabditis elegans. However, 0.5 mg·mL^-1 peptide of Poecilobdella manillensis significantly reduced ROS levels in Caenorhabditis elegans; 0.5 and 1.0 mg·mL^-1 peptide of Poecilobdella manillensis significantly reduced lipofuscin levels in Caenorhabditis elegans. Peptide of Poecilobdella manillensis exhibits effective whitening and anti-aging activities, potentially mediated by its inhibition of tyrosinase activity and antioxidant effects.

Inflammation caused by chronic liver is primarily responsible for the occurrence and pathological progression of liver fibrosis. In the process of liver fibrosis, a large number of activated inflammatory signals promote the transformation of hepatic stellate cells (HSC) into myofibroblasts (MF), which eventually leads to the massive secretion and deposition of extracellular matrix (ECM) and the formation of scar tissue in the liver. To provide literature references for clinical diagnosis and treatment, this paper reviews the roles of HSC, Kupffer cells (KC), inflammasomes and inflammatory signaling in liver fibrosis.

Objective:To investigate the effects of IL-28B in a mouse model of dextran sulfate sodium (DSS)-induced colitis and to analyze the possible mechanism.Methods:Thirty-five male C57BL/6 mice were randomly divided into the following groups with seven mice in each group: control group, DSS group and three IL-28B groups (1.25 μg, 2.5 μg and 5 μg). The mice in the DSS group and IL-28B groups were fed with 2.5% DSS solution and from day 3, the IL-28B groups were given intraperitoneal injection of corresponding IL-28B every day and the DSS group was treated with PBS. During the experiment, the disease activity index (DAI) was evaluated daily. On day 8, the mice were sacrificed and peripheral blood, spleen, mesenteric lymph node and colon samples were collected. The colon samples were observed, measured in length and stained with HE, and histopathological scores were calculated based on HE staining. Changes of immune cells in different samples were detected by flow cytometry. ELISA was used to detect the expression of IL-12, IL-10, IL-1β, IL-6, IL-4 and IL-13 in serum and colon tissues.Results:Compared with the DSS group, the IL-28B group (2.5 μg) had lower DAI scores [(9.40±1.67) vs (3.50±1.73), P<0.01], less shortening of the colon [(5.16±0.61) cm vs (6.91±0.60) cm, P<0.01] and significantly lower histopathological scores [(7.33±0.58) vs (4.33±0.58), P<0.01]. Moreover, compared with the DSS group, the IL-28B group (2.5 μg) showed decreased macrophages in the peripheral blood [(21.39±3.21)% vs (15.63±2.98)%, P<0.05] and spleen [(3.03±0.28)% vs (2.05±0.48)%, P<0.05], and significantly increased mean fluorescence intensity of M2 macrophages in the colon [(1 361.00±293.40) vs (2 074.00±87.61), P<0.05]. IL-12 expression in colon tissues and IL-1β expression in serum were reduced, and IL-10, IL-4 and IL-13 expression in colon tissues was significantly increased in the IL-28B group (2.5 μg) as compared with those in the DSS group [IL-12: (31.72±6.92) pg/mg vs (5.41±3.41) pg/mg; IL-1β: (48.01±16.13) pg/ml vs (12.27±6.26) pg/ml; IL-10: (184.70±46.82) pg/mg vs (444.30±157.80) pg/mg; IL-4: (2.23±0.27) pg/mg vs (3.64±0.80) pg/mg; IL-13: (11.79±0.99) pg/mg vs (22.59±1.92) pg/mg; all P<0.05]. Conclusions:IL-28B might alleviate the severity of acute enteritis in mice by increasing the secretion of IL-4 and IL-13, regulating macrophage differentiation and modulating the expression of inflammatory factors.

OBJECTIVE To study different extraction fractions of Agrimonia pilosa against h epatic fibrosis. METHODS Using hepatic stellate cells HSC-T 6 of rats as objects ，the effects of different extraction fractions （total extract ，ethyl acetate fraction ， petroleum ether fraction and n-butanol fraction ）with different concentrations （0.5，5，50，500，5 000 μg/mL，calculated by raw drug）of A. pilosa on the proliferation of HSC-T 6 cells were detected （after treated for 24，48，72 h）；median inhibition concentration（IC50）was also caculated. Platelet-derived growth factor （PDGF-BB）was used to induce the activation of HSC-T 6 cells to establish hepatic fibrosis cell model. Flow cytometry was used to detect the effects of different extraction fractions of A. pilosa on apoptosis of HSC-T 6 cells. The expression of collagen Ⅰ（Col-Ⅰ）in the supernatant was detected by enzyme linked immunosorbent assay. The expressions of α-smooth muscle actin （α-SMA），Col-Ⅰ，B-cell lymphoma- 2（Bcl-2），Bcl-2-associated X protein （Bax）and caspase- 3 were detected by Western blot assay. RESULTS Total extract ，ethyl acetate fraction ，petroleum ether fraction and n-butanol fraction of A. pilosa could significantly increase the apoptotic rate of HSC-T 6 cells（P＜0.01）. After treated for 24 h，IC50 of above fractions were 50.17，20.75，5.82，4.09 μg/mL，respectively. After intervened with PDGF-BB ，the expression of Col- Ⅰ in supernatant of HSC-T 6 cells as well as protein expression of Col- Ⅰ，α-SMA，Bcl-2，Bax and caspase- 3 in HSC-T6 cells were increased significantly （P＜0.01）. After intervened with different extraction fractions of A. pilosa ，most of the expressions of above proteins in HSC-T 6 cell culture supernatant or cells were significantly reversed compared with PDGF-BB group （P＜0.05 or P＜0.01）， and the intervention effect of n-butanol fraction of A. pilosa was the most significant. CONCLUSIONS Different extraction fractions of A. pilosa can inhibite the proliferation of HSC-T 6 cells and induce their apoptosis；n-butanol fraction from A. pilosa may be an effective fraction to exert the effect of anti-hepatic fibrosis.

Objective:To explore the clinical characteristics and risk factors of influenza virus complicated with gram-positive bacterial infection in children.Methods:The clinical data of children with influenza virus complicated with gram-positive bacterial infection hospitalized at Shenzhen Children′s Hospital affiliated to China Medical University from January 2013 to December 2019 (observation group) were retrospectively studied.During the same period, 110 hospitalized children with influenza virus infection without co-infection were selected as the control group.The clinical data of the children in two groups were analyzed.Logistic regression analysis was used to analyze the risk factors of influenza virus complicated with gram-positive bacterial infection.Results:There were 108 children in the observation group, including 68 boys and 40 girls, with the age of(2.6±1.8)years, and 100(92.6%) children under 5 years old.Incidence month distribution: 61 cases from January to March, 15 cases from April to June, 13 cases from July to September, and 19 cases from October to December.In the observation group, 73 cases were infected with influenza A virus, 35 cases were infected with influenza B virus, 94(87.0%)cases were complicated with Streptococcus pneumoniae infection, 11 cases with Group A Streptococcus infection and 8 cases with Staphylococcus aureus infection.And 15 (13.9%) cases had underlying diseases.None of the patients in the observation group received pneumococcal conjugate vaccine, and two cases received influenza vaccine within one year.There were 110 children in the control group, including 57 boys and 53 girls, with the age of (5.0±2.4)years old.There were 80 cases of influenza A virus infection and 30 cases of influenza B virus infection.Four cases had underlying diseases, six cases received 13-valent pneumococcal conjugate vaccine and 12 cases received influenza vaccine within one year.Compared with the control group, the children in the observation group were younger[(2.6±1.8)years vs.(5.0±2.4) years, χ2=-7.935, P<0.001], had more underlying diseases[13.9%(15/108)vs.3.6%(4/110), χ2=7.200, P=0.007], less proportion of influenza vaccine[1.9%(2/108)vs.10.9%(12/110), χ2=7.439, P=0.006], the hospitalization time was longer[6(5, 7)d vs.4(3, 5)d, Z=-7.278, P<0.001], and mone cases of first use of neuraminidase inhibitors(NAI) for more than 48 hours[75.9%(82/108)vs.14.5%(16/110), χ2=82.971, P<0.001]. In the observation group, there were 97 culture-positive specimens of Streptococcus pneumoniae, including 89 of sputum/bronchoalveolar lavage fluid, five of blood culture and three of cerebrospinal fluid.All Streptococcus pneumoniae were resistant to erythromycin and clindamycin; the resistance rates of non-meningitis Streptococcus pneumoniae to ceftriaxone, cefotaxime and penicillin were 7.7%, 5.5% and 1.1%, respectively, and all the strains were sensitive to vancomycin, linezolid and levofloxacin.All patients in the observation group were treated with NAI and antibiotics, 37 cases were treated with bronchoalveolar lavage, 27 cases were admitted to pediatric intensive care unit, 10 cases were treated by non-invasive continuous positive airway pressure ventilation, and 17 cases received mechanical ventilation; 6 cases died.Logistic regression analysis showed that underlying diseases, unvaccinated with influenza and (or) pneumococcal vaccine, and the first use of NAI>48 hours were risk factors for influenza virus complicated with gram-positive bacterial infection. Conclusion:Influenza virus complicated with gram-positive bacterial infection can aggravate the illness and even death of children.Early identification of gram-positive bacterial infection, timely treatment of NAI and antibiotics, and active control of complications could be helpful to improve the cure rate.Strengthening influenza and pneumococcal vaccine during flu season can help reduce infection.

Background and Objectives: Human umbilical cord mesenchymal stem cells (HUC-MSCs) are promising candidates for cell-based therapy in regenerative medicine or other diseases due to their superior characteristics, including higher proliferation, faster self-renewal ability, lower immunogenicity, a noninvasive harvest procedure, easy expansion in vitro, and ethical access, compared with stem cells from other sources. Methods: and Results: In the present study, we knocked down the expression of SOX9 in HUC-MSCs by lentivirus interference and found that knockdown of SOX9 inhibited the proliferation and migration of HUC-MSCs and influenced the expression of cytokines (IL-6 and IL-8), growth factors (GM-CSF and VEGF) and stemness-related genes (OCT4 and SALL4). In addition, the repair effect of skin with burn injury in rats treated with HUC-MSCs transfected with sh-control was better than that rats treated with HUC-MSCs transfected with shSOX9 or PBS, and the accessory structures of the skin, including hair follicles and glands, were greater than those in the other groups. We found that knockdown of the expression of SOX9 obviously inhibited the expression of Ki67, CK14 and CK18. Conclusions In conclusion, this study will provide a guide for modifying HUC-MSCs by bioengineering technology in the future.

Objective:To study the clinical value of blood neutrophil gelatinase-associated lipocalin (NGAL) in the early diagnosis and prognostic evaluation of late-onset sepsis in very/extremely low birth weight infants (VLBWI/ELBWI).Method:From January 2017 to December 2019, VLBWI/ELBWI older than 3 days admitted to NICU of our hospital were prospectively enrolled in the study. The infants were assigned into suspected-sepsis group and non-infection (control) group according to their clinical symptoms and laboratory indicators. In the suspected-sepsis group, complete blood count, C-reactive protein (CRP), procalcitonin (PCT) and blood culture were examined on the 1st day of disease onset and blood NGAL was examined on the 1st day of disease onset, 3rd day of treatment and 2nd week of treatment. In the control group, blood NGAL was examined at the time of enrollment. The suspected-sepsis group was later assigned into sepsis group and non-sepsis infection group and the sepsis group was further assigned into mild sepsis group and severe sepsis group according to the severity of the disease. Blood NGAL levels between the sepsis group and the non-sepsis infection group on the 1st day of onset and the control group were compared. The dynamic changes of NGAL in the sepsis group and the non-sepsis infection group at different time points were compared and analyzed. ROC curve of NGAL level on the first day of onset predicting sepsis was drawn.Result:(1) On the 1st day of disease, the sepsis group (n=106) had higher level of NGAL compared with non-sepsis infection group (n=121) and the control group (n=84). Non-sepsis infection group had significantly higher level of NGAL compared with the control group ( P<0.05). (2) A gradual decrease of NGAL was found in both sepsis and non-sepsis infection group. Significantly higher level of NGAL in sepsis group was found comparing with non-sepsis infection group at different time points ( P<0.05). (3) For blood culture positive and negative patients in the sepsis group, no statistically significant differences existed in NGAL,CRP, PCT levels on the 1st day of disease onset ( P>0.05).(4) The NGAL level in the severe sepsis group was significantly higher than the mild sepsis group on the 1st day of disease onset ( P<0.05). However,CRP and PCT showed no differences between the two groups. (5) On the 1st day of disease onset, to establish the diagnosis of sepsis, the area under the ROC curve of NGAL level was 0.852. The sensitivity and specificity of cut-off value 205.25 ng/ml were 84.0% and 66.9%, respectively. Conclusion:The serum NGAL level is elevated in VLBWI/ELBWI with late-onset sepsis. The more severe the sepsis,the more elevated the NGAL level. NGAL has certain predictive value for late onset sepsis in VLBWI/ELBWI.

Objective: To investigate the role of CD11b agonist leukadherin-1 (LA1) in the development of intestinal inflammation and colitis disease in a mouse model of dextran sulfate sodium (DSS)-induced colitis. Methods: The mouse model of experimental colitis was induced by DSS. Body weight changes and survival status were monitored every day. The length of colons was measured at day 7. Colon tissue sections were stained with hematoxylin and eosin (HE) and observed under an optical microscope for pathological analysis. The percentages of apoptotic cells in colon tissues were observed by TUNEL staining. Myeloperoxidase (MPO) activity was measured with MPO activity detection kit. IL-1β and TNF-α levels were detected by ELISA. Macrophages and TNF-α in colon tissues were observed using immunofluorescence staining and confocal microscopy. Flow cytometry was performed to detect the changes in TLR4 expression on macrophages after stimulating mice with LA1 for 0, 3, 6 and 12 h. Moreover, TLR4 expression was also measured by Western blot after treating bone marrow-derived macrophages (BMDMs) with LA1 for 0, 3, 6 and 12 h. Unpaired t-test was used for statistical analysis. Results: Compared with the DSS group, the LA1+ DSS group presented lower mortality rate, greater body weight and longer colon and the differences between the two groups were statistically significant. Moreover, the LA1+ DSS group showed lighter pathological damages, decreased percentage of apoptotic cells and suppressed MPO activity as compared with those of the DSS group. The number of macrophages and the relative concentrations of IL-1β and TNF-α in colon tissues were lower in the LA1+ DSS group than in the DSS group, and the differences between the two groups were statistically significant. There was no significant difference in the total expression of TLR4 on macrophages before and after LA1 treatment. However, the mean flourscence indensity (MFI) of TLR4 was weaker on the LA1-treated macrophages than on the untreated macrophages. Conclusions LA1 could alleviate the DSS-induced experimental colitis in mice through suppressing the activation of TLR4 pathway on macrophages. This study provided a new insight and theoretical reference for understanding the pathogenesis of inflammatory bowel diseases.

Objective To investigate the dynamic changes of soluble urokinase-type plasminogen activator receptor (suPAR) and its predictive value in late-onset sepsis in the newborn.Method To collect the data of neonates aged 7 days and older,who were diagonsed to have infections.They were admitted to neonatal intensive care unit of our Hospital from January 2014 to January 2015.The group of sepsis and nonseptic group were assigned according to the diagnostic criteria of sepsis,and a control group was selected without infection.Blood cultures were collected in patients on the first day when infection was identified and the serum suPAR and CRP were measured on the first day,fourth day and tenth day respectively.The controls were tested with suPAR and CRP when infection was excluded.The levels of blood suPAR and CRP in the three groups were compared and the receiver-operating characteristic curve was performed according to serum suPAR level of neonates with sepsis on the first day.Result A total of 65 infants with infections (40 were septic and 25 were non-septic) were enrolled in this study and 20 patients were selected as control group.There were significant differences in serum suPAR and CRP levels between the patients with and without infection (P ＜ 0.001).The level of suPAR in the survivors of the sepsis group was significantly decreased as time went by,and the difference was statistically significant on the 10th day compared with the 1 st day [9.3 (8.2,13.1) ng/ml vs.18.9 (14.8,24.7) ng/ml,P ＜ 0.05].The level of CRP increased first initially and then decreased with time,while the highest level was on the 4th day and the difference was statistically significant compared with the 10th day [19.0 （ 6.8,56.4) mg/L vs.6.4 (2.5,12.0) mg/L,P ＜ 0.05].The levels of serum suPAR and CRP in non-sepsis group were not significantly different (P ＞ 0.05).There were no deaths in the sepsis group and the non-septic group,but the levels of suPAR between survivals and deaths in the infection groups were statistically significant [15.4(10.6,21.6) ng/ml vs.22.6 (15.4,31.9) ng/ml,Z =-2.063,P =0.039].The area under the receiver-operating characteristic curve of serum suPAR was 0.955 (95％ CI 0.906 ～ 1.000,P ＜0.001),and the sensitivity was 90％ and the specificity was 100％ when the suPAR level was 10.9 ng/ml.Conclusion Early elevated serum suPAR levels were prominently related to the severity of neonatal late-onset sepsis.The level of first day suPAR has a high sensitivity and specificity in the prognosis of sepsis and can be helpful to predict the prognosis.

Objective To investigate the traditional Chinese medicine(TCM) syndromes distribution rule of nonalcoholic fatty liver disease(NAFLD) and its correlation with related clinical indexes.Methods The general condition,TCM four diagnostic methods,biochemical and CT results in 1950 cases of NAFLD in Chongqing City were investigated for analyzing the TCM syndromes distribution rule and its correlation with biochemistry and CT.Results In 1950 cases,the accumulation and binding of damp-heat,congestion of dampness turbidity,stagnation of liver-QI with spleen deficiency,intermin-gled phlegm and blood stasis and yin deficiency of both liver and kidney accounted for 36.62 ％,27.69 ％,19.38 ％,10.10 ％ and 6.21 ％ respectively;there was statistically significant difference in age among different TCM syndromes(P＜0.05);the vin deficiency syndrome of both liver and kidney and intermin-gled phlegm and blood stasis in severe fatty liver were maximal;glutamic-pyruvic transaminase(ALT) and glutamic-oxalacetic transaminase(AST) level was higher in the accumulation and binding of damp-heat;the level of fasting plasma gluco se(FBG) was higher in the yin deficiency syndrome of both liver and kidney;the total cholesterol(TC),triglyceride(TG) and FBG levels were lower in the stagnation of liver-QI with spleen deficiency,the differences were statistically significant(P＜0.05).Conclusion In NAFLD patients,the accumulation and binding of damp-heat distribution is maximal,the proportion of severe fatty liver with vin deficiency syndrome of both liver and kidney is higher.Different dialectical types may play an important role in the clinical indexes and disease development.