Aim To investigate the proliferative effect and the apoptosis of rat epididymal epithelial cells induced by podophyllotoxin and its underlying mechanisms.Methods Primary epididymal epithelial cells were cultured in vitro.CCK-8 assay was used to analyze proliferation of epididymal epithelial cells induced by podophyllotoxin on 24, 48 and 72 h.The ultra structural changes of the epididymal epithelial cells were observed by transmission electron microscope.AnnexinV-FITC/PI staining was used to quantify the percentages of apoptosis in the total cell population.The TdTmediated dUTP nick end labeling(TUNEL) technique was applied to observe the morphological changes of apoptotic cells.The expression of tumor necrosis factor alpha(TNF-α) mRNA was investigated by real-time RT-PCR.The level of TNF-α in cell culture supernatant was measured by enzyme-linked immunosorbent assay(ELISA) technology.Western blot was per-formed to determine the protein expression of cytochrome C, caspase-3, caspase-8, caspase-9.Results Podophyllotoxin significantly inhibited the activity of proliferation and induced apoptosis of epididymal epithelial cells in a dose-and time-dependent manner(P<0.05), with a 50％ inhibitory concentration(IC50) value and corresponding 95％ confidence intervals(CI) of 59.36(15.50～227.41), 0.37(0.080～1.70), 0.077(0.017～0.35) μmol·L-1 at 24, 48 and 72 h, respectively.Podophyllotoxin induced cell volume turned round and cell nuclear fragmentation and mitochondrial vacuolation.RT-PCR and ELISA results showed that podophyllotoxin improved the expression of TNF-α mRNA and protein.Western blot results demonstrated that podophyllotoxin activated the protein expression of cytochrome C, caspase-3, caspase-8 and caspase-9.Conclusion Podophyllotoxin can induce rat epididymal epithelial cell apoptosis through both the mitochondria-regulated intrinsic pathway and the TNF receptor-mediated extrinsic pathway.

Objective To establish RNase L gene knockout HEK 293 cell lines using CRISPR/Cas9 system.Methods Small guide RNA ( sgRNA) sequences of human RNase L were designed and sgRNAs were inserted into pCas-Guide and pCas-guide RNA(gRNA) vectors were obtained.The donor DNA sequences of the homologous arm were designed for RNase L knockout .In the presence of the right homologous arm , the resistance gene of hygromycin B and the left homologous arm as templates of homology-directed repair , the donor DNA template was amplified by overlopping PCR and cloned into the pBackZero-T expression vector and pBackZero-T-RNase LK vector was obtained .The pCas-gRNA vector and pBackZero-T-RNase LK vector were co-transfected into HEK293 cells to establish the stable expression cell line of RNase L gene knockout .Cells were cultured with hygromycin B , while Western blotting and DNA sequencing were used to analyze the gene of RNase L knockout from genome .Results and Conclusion The pCas-gRNA vector and pBackZero-T-RNase LK vector were successfully constructed.Five RNase L gene knockout HEK293 cell lines were generated,contributing to the study of the biological function and molecular mechanism of RNase L .

Objective To study the treatment of multilevel atherosclerotic occlusive disease of the lower(extremity).Methods From March 2004 to January 2006,intraoperative iliac balloon angioplasty and stenting combined with simultaneous vascular bypass and profundoplasty were performed on 24 limbs in 21 patients(suffering) from lower extremity multilevel atherosclerotic occlusive disease.In 24 limbs iliac balloon angioplasty and stent placement were performed with simultaneous profundoplasty in 12 limbs and femoro-popliteal bypass in 14 limbs.Results Surgical procedures were technically successful in all patients.There were no severe postoperative morbidity or mortality.Before-operative vs postoperative ABI was 0.24?0.13 vs 0.63?(0.18)(P

Objective:To explore the effects of human-derived fibrin glue on prevention of postoperative complications of endoscopic submucosal dissection (ESD) in early esophageal squamous cancer and precancerous lesions.Methods:A total of 210 patients with early esophageal squamous cancer or precancerous lesions who underwent ESD at Department of Gastroenterology, Zhongda Hospital Affiliated to Southeast University from April 2017 to April 2020 were included in this retrospective study. Seventy-three cases (79 esophageal lesions) where human-derived fibrin glue was used before retrieving endoscope were included in the observation group, while 137 cases (156 esophageal lesions) where fibrin glue was not used were included in the control group. The postoperative complications and pain were compared between the two groups.Results:Clinical data including general information, longitudinal length, Paris type, pathological type, invasion depth, circumferential range, area of resection, duration of operation and local steroid used were similar between the two groups ( P>0.05). The incidences of perforation, delayed bleeding and esophageal stenosis in the observation group were 2.7% (2/73), 1.4% (1/73), and 16.4% (12/73), respectively, and were 2.9% (4/137), 1.5% (2/137), and 13.1% (18/137), respectively in the control group. There were no significant differences between the two groups ( P>0.05). The incidence of postoperative pain in the observation group was 53.4% (39/73), which was significantly lower than that in the control group of 70.8% (97/137) ( χ2=6.302, P=0.012). The incidences of mild, moderate and severe pain in observation group on the day of ESD were 9.6% (7/73), 6.8% (5/73) and 5.5% (4/73), respectively, and 27.0% (37/137, χ2=8.724, P=0.003), 17.5% (24/137, χ2=4.554, P=0.033) and 0.7% (1/137, χ2=2.805, P=0.094), respectively in the control group. The incidences of mild, moderate and severe pain in the observation group on the first day after the operation were 26.0% (19/73), 5.5% (4/73) and 6.8% (5/73), respectively, and 29.2% (40/137, χ2=0.237, P=0.626), 14.6% (20/137, χ2=3.912, P=0.048) and 4.4% (6/137, χ2=0.193, P=0.660), respectively in the control group. The corresponding incidences on the second day after the operation were 5.5% (4/73), 0 and 1.4% (1/73) in the observation group and 19.0% (26/137, χ2=7.087, P=0.008), 2.9% (4/137) and 0 in the control group, respectively. Conclusion:Human-derived fibrin glue shows no obvious preventive effect on post-ESD bleeding, perforation or stenosis in early esophageal cancer and precancerous lesions. However, it can significantly reduce the incidence of ESD-related postoperative pain, especially the incidences of mild and moderate pain.

Objective:To compare the effects of citrate and heparin anticoagulation on coagulation function and efficacy in children with septic shock undergoing continuous blood purification (CBP), and to provide guidance for CBP anticoagulation in children with septic shock.Methods:A case control study was conducted. Thirty-seven children with septic shock admitted to the pediatric intensive care unit (PICU) of the First Affiliated Hospital of Gannan Medical University from July 2019 to September 2022 were enrolled as the research subjects. The patients were divided into citrate local anticoagulation group and heparin systemic anticoagulation group according to different anticoagulation methods. The baseline data, the level of coagulation indicators [prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (Fib), D-dimer] before treatment and 1 day after weaning from CBP, serum inflammatory mediators [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), hypersensitivity C-reactive protein (hs-CRP), procalcitonin (PCT)], bleeding complications during CBP and 7-day mortality were collected.Results:A total of 37 cases were enrolled finally, including 17 cases with citric acid local anticoagulation and 20 cases with heparin systemic anticoagulation. There was no statistically significant difference in general data such as gender, age, and body weight of children between the two groups. There were no statistically significant differences in baseline levels of coagulation indicators and inflammatory mediators before treatment of children between the two groups. One day after weaning from CBP, both groups showed varying degrees of improvement in coagulation indicators compared with those before treatment. Compared with before treatment, the PT of the heparin systemic anticoagulation group was significantly shortened after 1 day of weaning (s: 11.82±2.05 vs. 13.64±2.54), APTT and TT were significantly prolonged [APTT (s): 51.54±12.69 vs. 35.53±10.79, TT (s): 21.95±4.74 vs. 19.30±3.33], D-dimer level was significantly reduced (mg/L: 1.92±1.58 vs. 4.94±3.94), with statistically significant differences (all P < 0.05). While in the citrate local anticoagulation group, only APTT was significantly prolonged after treatment compared with that before treatment (s: 49.28±10.32 vs. 34.34±10.32, P < 0.05). There were no statistically significant differences in other coagulation indicators compared with before treatment. Compared with the citric acid local anticoagulation group, the PT of the heparin systemic anticoagulation group was significantly shortened after treatment (s: 11.82±2.05 vs. 13.61±3.05, P < 0.05), and the D-dimer level was significantly reduced (mg/L: 1.92±1.58 vs. 3.77±2.38, P < 0.01). The levels of inflammatory mediators in both groups were significantly reduced 1 day after CBP weaning compared with those before treatment [citric acid local anticoagulation group: hs-CRP (mg/L) was 12.53±5.44 vs. 22.65±7.27, PCT (μg/L) was 1.86±1.20 vs. 3.30±2.34, IL-6 (ng/L) was 148.48±34.83 vs. 202.32±48.62, TNF-α (ng/L) was 21.38±7.71 vs. 55.14±15.07; heparin systemic anticoagulation group: hs-CRP (mg/L) was 11.82±4.93 vs. 21.62±8.35, PCT (μg/L) was 1.90±1.08 vs. 3.18±1.97, IL-6 (ng/L) was 143.81±33.41 vs. 194.02±46.89, TNF-α (ng/L) was 22.44±8.17 vs. 56.17±16.92, all P < 0.05]. However, there was no statistically significant difference between the two groups (all P > 0.05). There was no statistically significant difference in bleeding complication during CBP and 7-day mortality in children between the citrate local anticoagulation group and the heparin systemic anticoagulation group (5.9% vs. 30.0%, 17.6% vs. 20.0%, both P > 0.05). Conclusions:Heparin for systemic anticoagulation and regional citrate anticoagulation can significantly reduce the levels of IL-6, TNF-α, hs-CRP and PCT in children with septic shock, and relieve inflammatory storm. Compared with citric acid local anticoagulation, heparin systemic anticoagulation can shorten the PT and reduce the level of D-dimer in children with septic shock, which may benefit in the prevention and treatment of disseminated intravascular coagulation (DIC).

Objective : To investigate the mechanism of miRNAs in glycyrrhizic acid treatment of alcohol⁃induced liver injury in rats . Methods : 45 male SD rats were randomly divided into glycyrrhizin group , model group and control group . The rats in glycyrrhizin group were given 56% liquor and glycyrrhizin , the rats in model group were given 56% liquor , and the rats in control group were given distilled water for 8 weeks . The blood was collected and the serum was separated by centrifugation to detect the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) . RNA were extracted from liver tissues , miRNAs were detected by rat miRNA microarray , and their expression levels were analyzed . The miRNA target genes of differential miRNA were predicted . Gene Ontology (GO) and KEGG Pathway enrichment analysis were used to understand the function of differential miRNA , and the differential miRNA⁃mRNA⁃Pathway regulatory network was constructed using Cytoscape to further screen the regulatory key miRNA and key pathways . qRT⁃PCR was used to verify the expression of selected miRNA . Results : Compared with the model group , the glycyrrhizin group could significantly improve the liver tissue lesions , and reduce the liver serum AST and ALT levels (P < 0. 05) . Compared the microarray data of glycyrrhizin group and model group , a total of 13 differentially expressed miRNA were screened ( P < 0. 05 , fold change ≥ 1 . 5) , of which 10 were up⁃regulated and 3 were down⁃regulated . The GO classification annotation of differential miRNA target genes showed that differential miRNA were related to cell adhesion , antioxidant activity , metabolic process , biological process regulation , cell killing , immune system and other functions . The KEGG Pathway analyling pathway , Hippo signaling pathway , PI3K⁃Akt signaling pathway , wnt signaling pathway , apoptosis and other signaling pathways might play an important regulatory role in the improvement of alcoholic liver injury by glycyrrhic acid . Conclusion This study established the miRNA expression profile of glycyrrhizin in the treatment of alcoholic liver injury in rats , suggested that miR⁃615 , miR⁃107 ⁃3p and miR⁃292⁃5p might play an important role in the treatment of alcoholic liver injury by glycyrrhizin .

Objective To establish an allogeneic rat model of endometriosis and to evaluate the effects of gonadotropin-releasing hormone (GnRH) agonist GenSci006 on experimental rat endometriosis. Methods Endometrium from SPF grade donor female SD rats were transplanted onto the abdominal wall of recipient female rats to construct an allogeneic endometriosis model. The rats undergoing sham surgery were divided into the sham group. Three weeks later, the length, width and height of the ectopic endometrium were measured, and the volume of the endometrium (V₁) was calculated before drug administration. The modeling rats were randomly divided into four groups: model group, triptorelin group (0.25 mg/kg), GenSci006-1 group (0.125 mg/kg) and GenSci006-2 group (0.25 mg/kg). Each group had 16 rats and received a single dose of the corresponding drug. The sham group and model group were administered an equal volume of solvent. Three weeks after administration, ectopic endometrium was measured to calculate the volume V₂ and inhibition rate. The effect of GenSci006 on rat uterus and ovarian tissues was assessed by comparing organ coefficients and changes in pathological sections. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of serum estradiol (E₂), progesterone (P₄), follicle stimulating hormone (FSH), and luteinizing hormone (LH). Real-time fluorescent quantitative PCR was used to detect the expression of GnRH receptor (GnRHR) mRNA in the hypothalamus and pituitary. Western blot was used to detect the expression of estradiol receptor alpha (ERα), beta (ERβ) and progesterone receptor (PR) in ectopic endometrium. Results Three weeks after administration, compared with the model group, the body weight of rats in the triptorelin and GenSci006-2 groups significantly increased (P < 0.05), while the volume of ectopic endometrium significantly decreased (P < 0.05). Compared with the sham group, the model group showed no significant changes in uterine and ovarian organ coefficients or endometrial thickness (P > 0.05). Compared with the model group, the uterine organ coefficients and endometrial thickness were significantly reduced in the triptorelin and GenSci006-2 groups (P < 0.05). Compared with the sham group, the serum levels of E₂, P₄, FSH and LH in the model group showed no significant changes (P > 0.05). Compared with the model group, the ovarian organ coefficient and serum P₄ levels of rats in the Triptorelin, GenSci006-1, and GenSci006-2 groups were significantly reduced (P < 0.05), while the serum LH levels of rats in the GenSci006-1 group were significantly increased (P < 0.05). However, there were no significant changes in serum E₂ and FSH levels in each group (P > 0.05). Compared with the model group, the expression levels of GnRHR mRNA in the pituitary tissue of rats in the triptorelin and GenSci006-2 groups were significantly downregulated (P < 0.05), with no significantly changes in the hypothalamus (P > 0.05). There were no significant changes in the expression level of GnRHR mRNA in the hypothalamus or the protein levels of ERα, ERβ and PR in the ectopic endometrial tissue in any group (P > 0.05). Conclusion The allogeneic endometriosis rat model is a suitable animal model for screening and evaluating drugs for treating endometriosis. The volume of ectopic endometrium, inhibition rate, uterine and ovarian organ coefficients, and serum E₂ levels may serve as indicators for detecting drug efficacy.