Objective To investigate the role of nitric oxide synthase(NOS)in the regulation of myocardial ischemia-reperfusion(IR)injury in ovariectomized(OVX)rats.Methods A total of 132 female SD rats were subjected,and 48 of them were randomly divided into sham operation group,IR group,OVX group and combined group,with 12 in each group.In order to explore the role of endothelous NOS(eNOS)and inducible NOS(iNOS)in ovariectomization increasing myo-cardial IR injury,another 84 mice were divided into negative sham group,negative IR group,nega-tive combined group,eNOS+IR group,eNOS combined group,iNOS small interfering RNA(si-iNOS)+IR group and si-iNOS combined group,with 12 in each group.The mice of the corre-sponding groups were injected with adeno-associated virus(AAV)overexpressing eNOS or knoc-king down iNOS via tail vein before OVX modeling.Myocardial infarct size,serum levels of lac-tate dehydrogenase(LDH)and creatine phosphokinase isoenzyme(CK-MB),LVEF,LVFS,and expression levels of eNOS and iNOS in the myocardial tissues were measured.Results The com-bined group had significantly increased level of iNOS in myocardium,larger myocardial infarct size and elevated serum LDH and CK-MB levels,but decreased myocardial expression of eNOS and LVEF and LVFS values than the IR group(P＜0.05).When compared with the negative combined group,the myocardial infarct size and serum LDH and CK-MB levels were decreased[(23.51±3.22)％and(26.21±2.93)％vs(58.78±5.42)％,(176.31±15.48 and 169.52±17.12 vs 328.85±37.12 U/L,35.41±6.41 and 34.77±5.94 vs 88.73±9.14 U/L,P＜0.05],and the LVEF and LVFS values were increased[(41.31±3.12)％and(42.09±3.41)％vs(30.77± 2.15)％,(21.47±1.57)％and(21.32±1.42)％vs(15.92±1.33)％,P＜0.05]in the eNOS com-bined group and si-iNOS combined group.Conclusion The decrease of eNOS expression and in-crease of iNOS expression are related to the aggravation of myocardial IR injury in OVX rats.

Objective To explore the protective mechanism of RNA binding protein LIN28 on diabetic nephropathy(DN).Methods LIN28 was overexpressed or knocked down by adenovirus in HEK 293T cells.The gene and functional signaling pathway significantly changed after overexpression of LIN28 were obtained through RNA Seq transcriptome sequencing and bioinformatics analysis.HEK 293T cells were divided into the Control group,the Treatment group,the Adv-LIN28-OE+D-ribose group and the Adv-LIN28-NC+D-ribose group in in vitro LIN28 overexpression studies.And HEK 293T cells were divided into the Control group,the Treatment group,the Adv-shRNA LIN28+D-ribose group and the Adv-shRNA NT+D-ribose group in in vitro LIN28 knockdown studies.ELISA was used to detect the levels of human advanced glycation end products(AGEs)of cells supernatants in the above groups.Western blot was used to detect the expression levels of RAGE,NF-κB and MMP-2 of cells in the above groups.Results The results of RNA-Seq transcriptome sequencing,GO analysis and KEGG analysis showed that overexpression of LIN28 resulted in a significant down-regulation of AGE-RAGE signaling pathway in HEK 293T cells(P<0.05).The results of ELISA showed that compared with the Control group,the cell in the Treatment group produced a large amount of AGEs(P<0.05);compared with the Treatment group,the AGEs level of cells in the Adv-LIN28-OE+D-ribose group was significantly decreased(P<0.05),while the AGEs level of cells in the Adv-shRNA LIN28+D-ribose group was increased(P>0.05).The results of Western blot showed that compared with the Control group,the expression levels of RAGE,NF-κB and MMP-2 of cells in the Treatment group were significantly increased(P<0.05);compared with the Treatment group,the expression levels of RAGE,NF-κB and MMP-2 of cells in the Adv-LIN28-OE+D-ribose group were significantly decreased(P<0.05),while the expression levels of RAGE,NF-κB and MMP-2 of cells in the Adv-shRNA LIN28+D-ribose group were significantly increased(P>0.05).Conclusion Overexpression of LIN28 by adenovirus at the cellular level in vitro can lead to significant differential expression of thousands of genes.In particular,it can inhibit the diabetes and complications-related AGE-RAGE signaling pathway,which is critical in the progression of DN disease,and play a role in protecting DN.

OBJECTIVE: To explore the underlying mechanism of inhibition by Jinkui Shenqi Pills (JKSQP) on glucocorticoid-enhanced axial length elongation in experimental lens-induced myopia (LIM) guinea pigs. METHODS: Sixty 2-week old male guinea pigs were randomly divided into 4 groups with 15 guinea pigs in each group, according to the random numbers generated by SPSS software: control, LIM, saline and JKSQP groups. The control group includes animals with no treatment, while the guinea pigs in the other 3 groups received lens-induced myopization on the right eyes throughout the experiment (for 8 weeks). The saline and JKSQP groups were given daily intraperitoneal injections of 10 mg/kg hydrocortisone for 2 consecutive weeks at the same time, and then orally administered either saline or JKSQP [13.5 g/(kg•d) for 6 consecutive weeks. Body weight, anal temperature and animal appearance were observed and recorded to evaluate the GC-associated symptoms. The ocular parameters, including refraction and axial length, were measured by streak retinoscopy and A-scan ultrasonography, respectively. The levels of plasma hormones associated with the hypothalamic-pituitary-adrenal axis (HPAA), including free triiodothyronine, free thyroxine, estradiol and testosterone, were measured by radioimmunoassay, and cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate were measured by enzyme-linked immunosorbent assay. In addition, the mRNA and protein expressions of retinal amphiregulin (AREG) was measured by quantitative real-time polymerase chain reaction and Western blotting, respectively. RESULTS: JKSQP effectively increased body weight and anal temperature, improved animal appearance and suppressed axial length elongation in glucocorticoid-enhanced myopic guinea pigs with normalization of 4 HPAA-associated plasma hormones (all P<0.05). The plasma level of cAMP was significantly increased, whereas the plasma level of cGMP and the mRNA and protein expressions of retinal AREG were decreased after treatment with JKSQP (all P<0.05). CONCLUSION JKSQP exhibited a significant inhibitory effect on axial length elongation with decreased expression of AREG in the retina, and normalized 4 HPAA-associated plasma hormones and the expression of cAMP and cGMP in GC-enhanced myopic guinea pigs.
Guinea Pigs ; Male ; Animals ; Glucocorticoids ; Hypothalamo-Hypophyseal System ; Pituitary-Adrenal System ; Myopia/metabolism* ; Body Weight ; RNA, Messenger ; Disease Models, Animal

OBJECTIVES: To investigate the incidence of extrauterine growth retardation (EUGR) and its risk factors in very preterm infants (VPIs) during hospitalization in China. METHODS: A prospective multicenter study was performed on the medical data of 2 514 VPIs who were hospitalized in the department of neonatology in 28 hospitals from 7 areas of China between September 2019 and December 2020. According to the presence or absence of EUGR based on the evaluation of body weight at the corrected gestational age of 36 weeks or at discharge, the VPIs were classified to two groups: EUGR group (n=1 189) and non-EUGR (n=1 325). The clinical features were compared between the two groups, and the incidence of EUGR and risk factors for EUGR were examined. RESULTS: The incidence of EUGR was 47.30% (1 189/2 514) evaluated by weight. The multivariate logistic regression analysis showed that higher weight growth velocity after regaining birth weight and higher cumulative calorie intake during the first week of hospitalization were protective factors against EUGR (P<0.05), while small-for-gestational-age birth, prolonged time to the initiation of total enteral feeding, prolonged cumulative fasting time, lower breast milk intake before starting human milk fortifiers, prolonged time to the initiation of full fortified feeding, and moderate-to-severe bronchopulmonary dysplasia were risk factors for EUGR (P<0.05). CONCLUSIONS It is crucial to reduce the incidence of EUGR by achieving total enteral feeding as early as possible, strengthening breastfeeding, increasing calorie intake in the first week after birth, improving the velocity of weight gain, and preventing moderate-severe bronchopulmonary dysplasia in VPIs.
Female ; Fetal Growth Retardation ; Gestational Age ; Hospitalization ; Humans ; Incidence ; Infant ; Infant, Newborn ; Infant, Premature ; Infant, Very Low Birth Weight ; Prospective Studies ; Risk Factors

Objective: To examine the clinical effect of minimally invasive duodenum preserving pancreatic head resection(DPPHR) for benign and pre-malignant lesions of pancreatic head. Methods: The clinical data of patients with diagnosis of benign or pre-malignant pancreatic head tumor were retrospectively collected and analyzed,all of them underwent laparoscopic or robotic DPPHR between October 2015 and September 2021 at Division of Gastrointestinal and Pancreatic surgery,Zhejiang Provincial People's Hospital. Thirty-three patients were enrolled with 10 males and 23 females. The age(M(IQR)) was 54(32) years old(range: 11 to 77 years old) and the body mass index was 21.9(2.9)kg/m²(range: 18.1 to 30.1 kg/m²). The presenting symptoms included abdominal pain(n=12), Whipple triad(n=2), and asymptomatic(n=19). There were 7 patients with hypertension and 1 patient with diabetes mellitus. There were 19 patients who were diagnosed as American Society of Anesthesiologists class Ⅰ and 14 patients who were diagnosed as class Ⅱ. The student t test,U test, χ² test or Fisher exact test was used to compare continuous data or categorized data,respectively. All the perioperative data and metabolic morbidity were analyzed and experiences on minimally invasive DPPHR were concluded. Results: Fourteen patients underwent laparoscopic DPPHR,while the rest of 19 patients received robotic DPPHR. Indocyanine green fluorescence imaging was used in 19 patients to guide operation. Five patients were performed pancreatico-gastrostomy and the rest 28 patients underwent pancreaticojejunostomy. Pathological outcomes confirmed 9 solid pseudo-papillary neoplasms, 9 intraductal papillary mucinous neoplasms, 7 serous cystic neoplasms, 6 pancreatic neuroendocrine tumors, 1 mucous cystic neoplasm, 1 chronic pancreatitis. The operative time was (309.4±50.3) minutes(range:180 to 420 minutes),and the blood loss was (97.9±48.3)ml(range:20 to 200 ml). Eighteen patients suffered from postoperative complications,including 3 patients experienced severe complications(Clavien-Dindo Grade ≥Ⅲ). Pancreatic fistula occurred in 16 patients,including 8 patients with biochemical leak,7 patients with grade B pancreatic fistula and 1 patient with grade C pancreatic fistula. No one suffered from the duodenal necrosis and none perioperative death was occurred. The length of hospital stay was 14(7) days (range:6 to 87 days). The follow-up was 22.6(24.5)months(range:2 to 74 months). None suffered from recurrence or metastasis. During the follow-up,all the patients were free of refractory cholangitis. Moreover,in the term of endocrine dysfunction,no postoperative new onset of diabetes mellitus were observed in the long-term follow-up. However,in the view of exocrine insufficiency,pancreatic exocrine insufficiency and non-alcoholic fatty liver disease (NAFLD) was complicated in 2 and 1 patient,respectively,with the supplement of pancreatic enzyme,steatorrhea and weight loss relieved,but NAFLD was awaited to be seen. Conclusions: Minimally invasive DPPHR is feasible and safe for benign or pre-malignant lesions of pancreatic head. Moreover,it is oncological equivalent to pancreaticoduodenectomy with preservation of metabolic function without refractory cholangitis.
Adolescent ; Adult ; Aged ; Child ; Duodenum/surgery* ; Female ; Humans ; Male ; Middle Aged ; Pancreas/surgery* ; Pancreatectomy ; Pancreatic Neoplasms/surgery* ; Pancreaticoduodenectomy ; Postoperative Complications ; Retrospective Studies ; Young Adult

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.
Animals ; Cell Differentiation ; Embryo, Mammalian/metabolism* ; Embryonic Development ; Mice ; Pluripotent Stem Cells/metabolism* ; RNA, Messenger/genetics* ; RNA, Ribosomal ; RNA-Binding Proteins/metabolism* ; Transcription Factors/metabolism* ; Zygote/metabolism*

Bitter almonds (Semen Armeniacae Amarum) are prone to oil deterioration during storage, so they often require mashing prior to clinical use. To confirm the medical value of bitter almonds "being mashed when used" and to determine the optimal storage conditions for bitter almonds, UPLC-MS/MS was used to perform a comparative study of the chemical composition of bitter almonds in different storage states (mashed and unmashed), storage times (0, 2 and 4 weeks), and storage temperatures (25 ℃ and 4 ℃). A total of 58 substances were identified in bitter almond extracts through literature review, this group's previous work, and a Compound Discoverer software search. Statistically significant differences were found in the chemical composition and content of bitter almonds in different storage states, storage times, and storage temperatures. The results show that the chemical composition of bitter almonds stored unmashed was more stable than that of bitter almonds stored mashed; the chemical composition of bitter almonds stored at 4 ℃ was more stable than that of bitter almonds stored at 25 ℃; and the shorter the storage time, the less the chemical composition changed. Amygdalin, the main medicinal component of bitter almonds, showed statistically significant differences in content under the above three storage conditions, which can be used as a potential quality marker for bitter almonds.

Using the concepts and methods of epigenetics and metabolomics, to investigate the overall action molecular mechanism of Chrysanthemi indici C (CIC), the anti-hepatitis B virus (HBV) active extracts from Flos chrysanthemi indici. The inhibitory effects of CIC on proliferation and hepatitis B surface antigen (HBsAg), hepatitis B envelope antigen (HBeAg) and HBV-DNA of HepG2.2.15 cells were detected by CCK-8 and antigen kit. The DNA methyltransferases (DNMTs)/ten-eleven-translocation-2 (TET2) equilibrium was detected by ELISA. Illumina 850K methylation chip, pyrosequencing and qPCR were used to determine the action pathway and target of CIC by GO and KEGG analysis. Cell metabolites were extracted with 80% methanol, and the changes of differential metabolites, differential metabolic pathways and cell microenvironment were detected by LC-MS and other metabolomics methods. The results showed that CIC could inhibit the proliferation, HBsAg, HBeAg and HBV-DNA of HepG2.2.15 cells obviously, down-regulate DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a) and DNA methyltransferase 3b (DNMT3b), up-regulate TET2, and restore the balance of DNMTs/TET2. The action targets of CIC were phospholipase C gamma 2 (PLCG2), phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2), 5-hydroxytryptamine receptor 2B (HTR2B), nerve growth factor (NGF), mainly involved in lipid metabolism, inflammation mediated regulation of transient receptor potential (TRP), phospholipase D signaling and advanced glycation end product-receptor for AGE (AGE-RAGE) signaling in diabetic complications pathways. CIC could significantly affect fatty acid metabolism and had great influence on phenolic acid, alkaloid and lipid metabolites in cell microenvironment. These results suggest that the action mechanism of CIC may be the synergistic action of multiple pathways and multiple targets, including related inflammatory pathways, immune pathways and lipid metabolism, through regulating epigenetic expression balance and restoring the balance of cell microenvironment.

Objective: To analyze the clinical epidemiological characteristics including composition of pathogens , clinical characteristics, and disease prognosis acute bacterial meningitis (ABM) in Chinese children. Methods: A retrospective analysis was performed on the clinical and laboratory data of 1 610 children <15 years of age with ABM in 33 tertiary hospitals in China from January 2019 to December 2020. Patients were divided into different groups according to age,<28 days group, 28 days to <3 months group, 3 months to <1 year group, 1-<5 years of age group, 5-<15 years of age group; etiology confirmed group and clinically diagnosed group according to etiology diagnosis. Non-numeric variables were analyzed with the Chi-square test or Fisher's exact test, while non-normal distrituction numeric variables were compared with nonparametric test. Results: Among 1 610 children with ABM, 955 were male and 650 were female (5 cases were not provided with gender information), and the age of onset was 1.5 (0.5, 5.5) months. There were 588 cases age from <28 days, 462 cases age from 28 days to <3 months, 302 cases age from 3 months to <1 year of age group, 156 cases in the 1-<5 years of age and 101 cases in the 5-<15 years of age. The detection rates were 38.8% (95/245) and 31.5% (70/222) of Escherichia coli and 27.8% (68/245) and 35.1% (78/222) of Streptococcus agalactiae in infants younger than 28 days of age and 28 days to 3 months of age; the detection rates of Streptococcus pneumonia, Escherichia coli, and Streptococcus agalactiae were 34.3% (61/178), 14.0% (25/178) and 13.5% (24/178) in the 3 months of age to <1 year of age group; the dominant pathogens were Streptococcus pneumoniae and the detection rate were 67.9% (74/109) and 44.4% (16/36) in the 1-<5 years of age and 5-<15 years of age . There were 9.7% (19/195) strains of Escherichia coli producing ultra-broad-spectrum β-lactamases. The positive rates of cerebrospinal fluid (CSF) culture and blood culture were 32.2% (515/1 598) and 25.0% (400/1 598), while 38.2% (126/330)and 25.3% (21/83) in CSF metagenomics next generation sequencing and Streptococcus pneumoniae antigen detection. There were 4.3% (32/790) cases of which CSF white blood cell counts were normal in etiology confirmed group. Among 1 610 children with ABM, main intracranial imaging complications were subdural effusion and (or) empyema in 349 cases (21.7%), hydrocephalus in 233 cases (14.5%), brain abscess in 178 cases (11.1%), and other cerebrovascular diseases, including encephalomalacia, cerebral infarction, and encephalatrophy, in 174 cases (10.8%). Among the 166 cases (10.3%) with unfavorable outcome, 32 cases (2.0%) died among whom 24 cases died before 1 year of age, and 37 cases (2.3%) had recurrence among whom 25 cases had recurrence within 3 weeks. The incidences of subdural effusion and (or) empyema, brain abscess and ependymitis in the etiology confirmed group were significantly higher than those in the clinically diagnosed group (26.2% (207/790) vs. 17.3% (142/820), 13.0% (103/790) vs. 9.1% (75/820), 4.6% (36/790) vs. 2.7% (22/820), χ²=18.71, 6.20, 4.07, all P<0.05), but there was no significant difference in the unfavorable outcomes, mortility, and recurrence between these 2 groups (all P>0.05). Conclusions: The onset age of ABM in children is usually within 1 year of age, especially <3 months. The common pathogens in infants <3 months of age are Escherichia coli and Streptococcus agalactiae, and the dominant pathogen in infant ≥3 months is Streptococcus pneumoniae. Subdural effusion and (or) empyema and hydrocephalus are common complications. ABM should not be excluded even if CSF white blood cell counts is within normal range. Standardized bacteriological examination should be paid more attention to increase the pathogenic detection rate. Non-culture CSF detection methods may facilitate the pathogenic diagnosis.
Adolescent ; Brain Abscess ; Child ; Child, Preschool ; Escherichia coli ; Female ; Humans ; Hydrocephalus ; Infant ; Infant, Newborn ; Male ; Meningitis, Bacterial/epidemiology* ; Retrospective Studies ; Streptococcus agalactiae ; Streptococcus pneumoniae ; Subdural Effusion ; beta-Lactamases

Objective To establish a new patient-derived xenograft (PDX) model of human liver cancer by inoculating the complex of human primary liver cancer cells and a novel microcarrier (microcarrier 6) into mice with normal immune function. Methods Primary liver cancer cells were isolated and extracted from the fresh human liver cancer tissue of five patients and were then co-cultured with microcarrier 6 to construct a three-dimensional tumor cell culture model in vitro . According to the type of graft, 75 male C57BL/6 mice were divided into cell control group, microcarrier control group, and experimental group (each sample corresponded to three groups, with 15 groups in total and 5 mice in each group). The liver cancer cell-microcarrier complex was implanted into the mice by subcutaneous inoculation, and tumor formation time, tumor formation rate, and histopathological manifestations were observed. The Fisher's exact test was used for comparison of categorical data between two groups. Results As for the liver cancer cells from the five patients, tumor formation was observed in the mice corresponding to three patients. In these three experiments, tumor formation was not observed in the control groups and was only observed in the experimental groups, and 12 of the 15 mice in the experimental groups had successful tumor formation, with a tumor formation rate as high as 80%, which was significantly different from that in the cell control groups and the microcarrier control groups (all P < 0.05). The tumor formation time was 5-7 days; the xenograft tumor grew rapidly, and HE staining showed nested or flaky cells with obvious heteromorphism, with the presence of pathological mitosis; immunohistochemical staining showed positive CK8/18, Hep, and Gpc-3, which was in accordance with the characteristics of human liver cancer cells. Conclusion This experiment successfully establishes a new PDX model of human liver cancer based on the complex of microcarrier 6 and human primary liver cancer cells in mice with normal immunity. This model can be used to better elucidate the mechanism of the development and progression of liver cancer in the body with normal immunity, and besides, it also provides a new animal model with higher value for the precise treatment of liver cancer.