Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.

ObjectiveOur recent study has demonstrated that extracellular acidification promotes lipid accumulation in macrophages via the activation of acid sensing ion channel 1 (ASIC1), but the underlying mechanism remains unclear. This study aims to explore the effect of extracellular acidification on macrophage lipophagy and the underlying mechanism. MethodsRAW264.7 macrophages were incubated with 25 mg/Lox-LDL in a pH 6.5 culture medium for 24 h to build macrophage-derived foam cell models induced by extracellular acidification. Then, RAW264.7 macrophages were cultured in the acidic medium of pH 6.5 with or without PcTx-1 (ASIC1 specific blocker, 10 μg/L) or Nec-1 (RIP1 specific inhibitor, 20 μmol/L) for 24 h, intracellular lipid accumulation was observed by oil red O staining. The expressions of total ASIC1, plasma membrane ASIC1, RIP1, p-RIP1 Ser166, TFEB, p-TFEB Ser142, LC3 and p62 were measured by Western blot. The co-localization of lipids (indicated by Bodipy) with LC3II (autophagosomes) and LAMP1 (lysosomes) was analyzed by a confocal laser scanning microscopy, respectively. Morphological changes of lipophagy in the cells were observed by using transmission electron microscopy. ABCA1-mediated cholesterol efflux was determined by cholesterol fluorescence kits. ResultsCompared with pH 7.4+ox-LDL group, the intracellular lipid accumulation in the pH 6.5+ox-LDL group was significantly increased. Meanwhile, the expressions of plasma membrane ASIC1, p-RIP1 Ser166, p-TFEB Ser142, and p62 proteins were elevated significantly, while LC3II protein level and LC3II/LC3I ratio were decreased. Accordingly, compared with pH 7.4+ox-LDL group, the macrophage lipophagy of the pH 6.5+ox-LDL group was inhibited as indicated by the decreased localization of lipid droplets with LC3 and LAMP1, a decrease in the number of lipophagosomes as well as an increase in lipid droplets. Furthermore, ATP binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux from the macrophages of pH 6.5+ox-LDL group reduced dramatically. However, these above effects of extracellular acidification on RAW264.7 macrophages were abolished by PcTx-1 and Nec-1, respectively. ConclusionThese findings suggest extracellular acidification promotes the phosphorylation of TFEB at Ser142 via activating ASIC1/RIP1 pathway, thereby impeding lipophagy in RAW 264.7 macrophages, and that ASIC1 may be a new potential target for preventing aberrant lipid accumulation diseases including atherosclerosis.

Objective To investigate the effect of Xinma Granules on respiratory mucosal immune function in chronic asthmatic mice.Methods Fifty female BALB/C mice were randomly divided into normal group,model group,low-dose Xinma Granules group,high-dose Xinma Granules group and Dexamethasone group,with 10 mice in each group.Except for the normal group,the mice in the other groups were sensitized and challenged with ovalbumin(OVA)to establish a chronic asthma model.After corresponding treatment,the levels of secretory immunoglobulin A(sIgA)and immunoglobulin E(IgE)in bronchial lavage fluid were measured by enzyme-linked immunosorbent assay(ELISA).The pathological changes of lung tissue were observed by hematoxylin-eosin(HE)staining.The expression of E-cadherin in lung tissue was detected by Western Blot.Results HE staining showed obvious airway inflammation in asthmatic mice.The concentration of sIgA in the bronchial lavage fluid of the model group was lower than that of the normal group(P＜0.05);the concentration of sIgA in the high-dose and the low-dose of Xinma Granules groups and the Dexamethasone group was higher than that in the model group(P＜0.05);the concentration of IgE in bronchial lavage fluid of model group was higher than that of normal group(P＜0.05);the concentration of IgE in the high-dose and the low-dose Xinma Granules groups and the Dexamethasone group was lower than that in the model group(P＜0.05).The relative expression of E-cadherin protein in lung tissue of the model group was lower than that of the normal group(P＜0.05);the relative expression of E-cadherin protein in lung tissue of mice in the high-dose and low-dose Xinma Granules groups and Dexamethasone group was higher than that in model group(P＜0.05).The improvement effect on above various indexes in high-dose Xinma Granules group and Dexamethasone group were superior to that in low-dose Xinma Granules group(P＜0.05),the differences between the both groups were statistically insignificant(P＞0.05).Conclusion Xinma Granules may improve the airway mucosal immune function of asthmatic mice by improving airway inflammation,increasing the concentration of sIgA in the respiratory tract,and enhancing the expression of E-cadherin protein in the respiratory tract.

Aim To investigate the effect of recombi-nant glycoprotein hormone β5/α2(rCGH)on lipolysis in 3T3-L1 adipocytes,and explore the underlying mechanism.Methods 3T3-L1 preadipocytes were cultured and induced to differentiate into mature adipo-cytes,then treated with different concentrations of rCGH for 24 h in vitro.Cell viability of 3T3-L1 adipo-cytes was evaluated by CCK-8 assay,the levels of in-tracellular triglyceride(TG)and glycerol in the culture supernatant were measured by enzymatic method,and the changes of lipid droplets were observed by oil red O staining.The expression levels of HSL and ATGL lipo-lytic proteins in adipocytes were detected by Western blot.To carry out the intervention experiment with dif-ferent concentrations of rCGH with or without the PKA inhibitor,H89,on the mature 3T3-L1 adipocytes,the cultured cells were divided into the control group,H89 pre treatment group,1 μmol·L-1 rCGH group,and(1 μmol·L-1 rCGH+H89)combined intervention group.The contents of intracellular TG and free glycer-ol were measured by enzymatic method,and the ex-pression of CREB and lipolysis-related proteins was de-tected using Western blot.Results Different concen-trations of rCGH(0.25,0.5,1,and 2 μmol·L-1)had no significant effect on the cell viability of adipo-cytes(P＞0.05).Compared with the control group,the treatment with rCGH significantly decreased the size of lipid droplets and intracellular TG content,while significantly elevated glycerol concentration in cell supernatant.rCGH treatment also stimulated the protein expression of p-HSL,ATGL,and p-PKA.In addition,the addition of a PKA inhibitor,H89,atten-uated the effects of rCGH on free glycerol level,intra-cellular TG content,and the expression of p-HSL,p-PLIN1,and p-CREB.Conclusions rCGH enhances the lipolysis of 3T3-L1 adipocytes by up-regulating the activities of HSL,ATGL and PKA,promoting glycerol release,inhibiting TG synthesis and lipid accumula-tion,and its mechanism of action is related to the acti-vation of cAMP/PKA/CREB signaling pathway.

AIM To study the secondary metabolites from the endophytic fungi Candida sp.of Berberis atrocarpa Schneid.METHODS The ethyl acetate fraction and petroleum ether fraction from the secondary metabolites of Candida sp.fermentation extract were separated and purified by silica gel,Sephadex LH-20 and preparative liquid chromatography,then the structures of obtained compounds were identified by physicochemical properties and spectral data.RESULTS Eighteen compounds were isolated and identified as 1-phenyl-1,2-ethanediol(1),4-hydroxyphenethyl alcohol(2),4-hydroxybenzoic acid(3),4-hydroxyphenylacetic acid(4),3-hydroxyphenylacetic acid(5),3-methylsulfinyl propionic acid(6),phenylacetic acid(7),(S)-N-nitroso-1-amino-p-hydroxy phenylethanol(8),2-phenylacetamide(9),p-hydroxybenzaldehyde(10),ethyl 2-(4-hydroxyphenyl)acetate(11),dibutyl phthalate(12),5,5'-dimethoxybiphenyl-2,2'-diol(13),3-indolealdehyde(14),N-acetyl-L-phenylalanine(15),9-hydroxy-10E,12Z-octadecadienoic acid(16),9-hydroxy-10E,12E-octadecadienoic acid(17),(6E)-5-methylene-6-tetradecenoic acid(18).CONCLUSION Compounds 1,3-8 and 10-18 are isolated from Candida sp for the first time.

AIM To explore the ameliorative effects of Zigui Yichong Formula on mitochondrial energy metabolism in in vitro model of premature ovarian insufficiency (POI).METHODS A POI model in vitro was established in human ovarian granulosa cell KGN with acrolein ( ACR ) for the further intervention of serum containing Zigui Yichong Formula.The control group,the ACR group,the Zigui Yichong Formula group,the EX527 ( SIRT1 inhibitor) group and the EX527+Zigui Yichong Formula group were set up.The cells had their apoptosis rate detected by flow cytometry;their ROS level detected by DCFH-DA method;their mitochondria number detected by Mito-Trakine fluorescence labeling method;their oxidative phosphorylation level of mitochondria detected by Seahorse method;their ATP level detected by colorimetry;their copy number of mtDNA and mRNA expressions of SIRT1,PGC-1α,Nrf1 and TFAM detected by RT-qPCR;and their protein expressions of SIRT1,PGC-1α,Ac-PGC-1α,Nrf1 and TFAM detected by Western blot.RESULTS Compared with the control group,the ACR group displayed increased apoptosis rate and ROS level of KGN cells ( P<0.05,P<0.01 );decreased number of mitochondria and mtDNA copy number ( P<0.01 );decreased basic value of mitochondrial aerobic respiration,maximum respiration,reserve value and ATP level (P<0.05,P<0.01),decreased mRNA and protein expressions of SIRT1,PGC-1α,Nrf1 and TFAM ( P<0.05,P<0.01 );and increased protein expression of Ac-PGC-1α( P<0.01 ).Compared with the ACR group,the Zigui Yichong Formula group demonstrated improvement in terms of the aforementioned indices levels ( P<0.05,P<0.01).The combination use of Zigui Yichong Formula and EX527 reversed the effects of single use of Zigui Yichong Formula on KGN cells ( P<0.05,P<0.01).CONCLUSION Zigui Yichong Formula may improve the mitochondrial energy metabolism disorder induced by ACR in KGN cells by increasing mitochondrial biosynthesis via SIRT1/PGC-1α signaling pathway.

AIM To explore the ameliorative effects of Zigui Yichong Formula on mitochondrial energy metabolism in in vitro model of premature ovarian insufficiency (POI).METHODS A POI model in vitro was established in human ovarian granulosa cell KGN with acrolein ( ACR ) for the further intervention of serum containing Zigui Yichong Formula.The control group,the ACR group,the Zigui Yichong Formula group,the EX527 ( SIRT1 inhibitor) group and the EX527+Zigui Yichong Formula group were set up.The cells had their apoptosis rate detected by flow cytometry;their ROS level detected by DCFH-DA method;their mitochondria number detected by Mito-Trakine fluorescence labeling method;their oxidative phosphorylation level of mitochondria detected by Seahorse method;their ATP level detected by colorimetry;their copy number of mtDNA and mRNA expressions of SIRT1,PGC-1α,Nrf1 and TFAM detected by RT-qPCR;and their protein expressions of SIRT1,PGC-1α,Ac-PGC-1α,Nrf1 and TFAM detected by Western blot.RESULTS Compared with the control group,the ACR group displayed increased apoptosis rate and ROS level of KGN cells ( P<0.05,P<0.01 );decreased number of mitochondria and mtDNA copy number ( P<0.01 );decreased basic value of mitochondrial aerobic respiration,maximum respiration,reserve value and ATP level (P<0.05,P<0.01),decreased mRNA and protein expressions of SIRT1,PGC-1α,Nrf1 and TFAM ( P<0.05,P<0.01 );and increased protein expression of Ac-PGC-1α( P<0.01 ).Compared with the ACR group,the Zigui Yichong Formula group demonstrated improvement in terms of the aforementioned indices levels ( P<0.05,P<0.01).The combination use of Zigui Yichong Formula and EX527 reversed the effects of single use of Zigui Yichong Formula on KGN cells ( P<0.05,P<0.01).CONCLUSION Zigui Yichong Formula may improve the mitochondrial energy metabolism disorder induced by ACR in KGN cells by increasing mitochondrial biosynthesis via SIRT1/PGC-1α signaling pathway.

Objective: To investigate the prognostic factors of children with congenital heart disease (CHD) who had undergone cardiopulmonary resuscitation (CPR) in pediatric intensive care unit (PICU) in China. Methods: From November 2017 to October 2018, this retrospective multi-center study was conducted in 11 hospitals in China. It contained data from 281 cases who had undergone CPR and all of the subjects were divided into CHD group and non-CHD group. The general condition, duration of CPR, epinephrine doses during resuscitation, recovery of spontaneous circulation (ROSC), discharge survival rate and pediatric cerebral performance category in viable children at discharge were compared. According to whether malignant arrhythmia is the direct cause of cardiopulmonary arrest or not, children in CHD and non-CHD groups were divided into 2 subgroups: arrhythmia and non-arrhythmia, and the ROSC and survival rate to discharge were compared. Data in both groups were analyzed by t-test, chi-square analysis or ANOVA, and logistic regression were used to analyze the prognostic factors for ROSC and survival to discharge after cardiac arrest (CA). Results: The incidence of CA in PICU was 3.2% (372/11 588), and the implementation rate of CPR was 75.5% (281/372). There were 144 males and 137 females with median age of 32.8 (5.6, 42.7) months in all 281 CPA cases who received CPR. CHD group had 56 cases while non-CHD had 225 cases, with the percentage of 19.9% (56/281) and 80.1% (225/281) respectively. The proportion of female in CHD group was 60.7% (34/56) which was higher than that in non-CHD group (45.8%, 103/225) (χ²=4.00, P=0.045). There were no differences in ROSC and rate of survival to discharge between the two groups (P>0.05). The ROSC rate of children with arthythmid in CHD group was 70.0% (28/40), higher than 6/16 for non-arrhythmic children (χ²=5.06, P=0.024). At discharge, the pediatric cerebral performance category scores (1-3 scores) of CHD and non-CHD child were 50.9% (26/51) and 44.9% (92/205) respectively. Logistic regression analysis indicated that the independent prognostic factors of ROSC and survival to discharge in children with CHD were CPR duration (odds ratio (OR)=0.95, 0.97; 95%CI: 0.92~0.97, 0.95~0.99; both P<0.05) and epinephrine dosage (OR=0.87 and 0.79, 95%CI: 0.76-1.00 and 0.69-0.89, respectively; both P<0.05). Conclusions: There is no difference between CHD and non-CHD children in ROSC and survival rate of survival to discharge was low. The epinephrine dosage and the duration of CPR are related to the ROSC and survival to discharge of children with CHD.
Cardiopulmonary Resuscitation ; Child ; Child, Preschool ; Female ; Heart Arrest/therapy* ; Heart Defects, Congenital/therapy* ; Humans ; Intensive Care Units, Pediatric ; Male ; Retrospective Studies

Objective:To analyze the antigenicity and genetic characteristics of two strains of Eurasian avian H1N1 swine influenza virus (EAS-H1N1) isolated from 2 human cases found in Gansu province to provide scientific reference for disease prevention and control.Methods:Two cases of human infection with EAS-H1N1 discovered in Gansu province since February 2021 were analyzed for antigenicity and genome comparison, and their genome characteristics were analyzed using software such as Mega7.0.Results:Two human infections with EAS-H1N1 had related environmental exposure history. Two strains were obtained: A/Gansu-Xifeng/1143/2021 and A/Gansu-Xifeng/1194/2021. The two strains of HA, NA genes were all from the Eurasian avian H1N1 swine influenza virus, and are closely related to the human infection EAS-H1N1 virus in Hebei and Tianjin and the EAS-H1N1 virus isolated from the mink in Shandong; the HA receptor binding sites of 2 strains E190D and D225E mutations occurred in all spots, and there were no H274Y and N294S mutations in NA protein.Conclusions:Some important molecular mutations occurred in the genome of people infected with EAS-H1N1 virus in Gansu province, which may lead to increased virulence and potential human-to-human transmission. Strengthening the research and analysis of EAS-H1N1 gene characteristics will help to monitor the mutation and mutation of the virus at the molecular level and scientifically prevent and control the epidemic.

Objective: To assess the risk of public health emergencies in Zhejiang Province, March 2021. Methods: An expert counsel was conducted to assess the risk of coronavirus disease 2019 ( COVID-19 ) , enteritis due to norovirus, chicken pox and influenza by professionals in Zhejiang CDC, based on the information from infectious disease and public health emergency surveillance in Zhejiang Province, domestic health administrative departments, World Health Organization, and European CDC. Results: In March 2021, the risk of imported COVID-19 epidemic will be high in Zhejiang Province, and the possibility of local spread could not be ruled out. The possibility of a large-scale outbreak of enteritis due to norovirus and a small-scale outbreak of chickenpox in schools and kindergartens could not be ruled out after the new term begins. An increased risk of influenza epidemic is predicted in collective units such as schools and kindergartens, yet the risk of a large-scale one will be low. Conclusion High attention should be paid to COVID-19 and enteritis due to norovirus, and general attention should be paid to chicken pox and influenza outbreak.