Objective To construct a uricase-deficient mouse model with stable inheritance using the CRISPR/Cas9 system,and evaluate its ability to simulate the disease characteristics of patients with hyperuricemia.Methods Double single guide RNAs(sgRNAs)were designed on both sides of exon 2～4 of the Uox gene.sgRNA and Cas9 mRNA for gene knockout were microinjected into the fertilized eggs of mice.After culture for 2～4 h,the embryos were transferred to surrogate mother mice to produce an F0 generation.Uox-knockout mice were identified by polymerase chain reaction and sequencing analysis.Positive mice were then mated with wild-type(WT)mice to produce an F1 generation,and heterozygous female and male F1 mice were then selected to obtain homozygous F2 mice.Serum and urine levels of uric acid,creatinine,and urea,and serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were detected and compared between homozygous and wild-type mice.Pathological changes in kidney and liver tissues were observed by hematoxylin and eosin and Masson staining.Results Urine levels of serum uric acid(male:(4116.8±1928.1)μmol/L,P＜0.001;female:(2998.0±547.7)μmol/L,P＜0.01)and serum levels of uric acid(male:(478.4±114.6)μmol/L,P＜0.001;female:(507.7±129.6)μmol/L,P＜0.001),creatinine((91.8±55.6)μmol/L,P＜0.001),urea((28.6±13.9)mmol/L,P＜0.05),ALT((53.3±23.3)U/L,P＜0.01),and AST((203.3±70.3)U/L,P＜0.001)were significantly increased in Uox-/-mice compared with WT mice.Histopathological examination showed moderate hepatocyte degeneration in the liver,moderate-to-severe tubular cystic dilation,degeneration,and fibrosis in the kidney,glomerular hypertrophy and hyperplasia,small-vessel dilation and congestion,and infiltration of stromal monocytes and lymphocytes in Uox-/-mice.Conclusions We successfully established a homozygous uricase-deficient mouse strain using CRISPR/Cas9 technology,as a suitable animal model for research in the field of hyperuricemia.

Objective To investigate the role of homocysteine(Hcy)in the pathogenesis of irritable bowel syn-drome(IBS)and its effects on intestinal motility,visceral hypersensitivity,and barrier function.Methods Clinical cohorts and animal models were combined for this study.Clinically,fifteen IBS patients meeting Rome Ⅲcriteria and 15 control individuals were enrolled to detect fecal Hcy levels and their correlation with symptoms.As for animal experiments,water avoidance stress(WAS)and 2,4,6-trinitrobenzene sulfonic acid(TNBS)chemical induction were utilized to establish IBS rat and mouse models,combined with a high-methionine diet(HMD)to simulate hyperhomocysteinemia.IBS symptoms were evaluated through fecal water content,carmine red intestinal transit time,and visceral hypersensitivity scores.Immune-fluorescence and Western blot were used to detect intes-tinal epithelial tight junction proteins.Serum and fecal Hcy concentrations were measured to assess Hcy levels.Statistical analyses included t-tests and One-way Anova.Results Fecal Hcy level in IBS patients were signifi-cantly higher than those in the healthy control group which demonstrated a positive correlation with defecation fre-quency(P＜0.01).In animal models,the combination of TNBS administration and a high-methionine diet markedly elevated serum and fecal Hcy levels in mice,while synergistically exacerbated intestinal motility disor-ders and visceral hypersensitivity.In vitro experiments showed that Hcy treatment down-regulates the expression of tight junction proteins in human colon cancer cell line(Caco-2).Conclusions Hcy plays an important role in the pathogenesis of IBS by impairing intestinal barrier function and enhancing visceral hypersensitivity,and it may serve as a potential new target for the treatment of IBS.

Objective To construct a uricase-deficient mouse model with stable inheritance using the CRISPR/Cas9 system,and evaluate its ability to simulate the disease characteristics of patients with hyperuricemia.Methods Double single guide RNAs(sgRNAs)were designed on both sides of exon 2～4 of the Uox gene.sgRNA and Cas9 mRNA for gene knockout were microinjected into the fertilized eggs of mice.After culture for 2～4 h,the embryos were transferred to surrogate mother mice to produce an F0 generation.Uox-knockout mice were identified by polymerase chain reaction and sequencing analysis.Positive mice were then mated with wild-type(WT)mice to produce an F1 generation,and heterozygous female and male F1 mice were then selected to obtain homozygous F2 mice.Serum and urine levels of uric acid,creatinine,and urea,and serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were detected and compared between homozygous and wild-type mice.Pathological changes in kidney and liver tissues were observed by hematoxylin and eosin and Masson staining.Results Urine levels of serum uric acid(male:(4116.8±1928.1)μmol/L,P＜0.001;female:(2998.0±547.7)μmol/L,P＜0.01)and serum levels of uric acid(male:(478.4±114.6)μmol/L,P＜0.001;female:(507.7±129.6)μmol/L,P＜0.001),creatinine((91.8±55.6)μmol/L,P＜0.001),urea((28.6±13.9)mmol/L,P＜0.05),ALT((53.3±23.3)U/L,P＜0.01),and AST((203.3±70.3)U/L,P＜0.001)were significantly increased in Uox-/-mice compared with WT mice.Histopathological examination showed moderate hepatocyte degeneration in the liver,moderate-to-severe tubular cystic dilation,degeneration,and fibrosis in the kidney,glomerular hypertrophy and hyperplasia,small-vessel dilation and congestion,and infiltration of stromal monocytes and lymphocytes in Uox-/-mice.Conclusions We successfully established a homozygous uricase-deficient mouse strain using CRISPR/Cas9 technology,as a suitable animal model for research in the field of hyperuricemia.

Eight new diterpenoids, Isodons A-H (1-8), comprising seco-abietane and abietane-type structures, together with 13 known analogues (9-21), were isolated from Isodon lophanthoides (Buch.-Ham. ex D. Don) Hara. The compounds (+)-3/(-)-3, (+)-4/(-)-4, and (+)-5/(-)-5 were identified as three enantiomeric pairs. The planar structures and absolute configurations of 1-8 were determined through high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1D & 2D nuclear magnetic resonance (NMR) spectroscopy, electronic circular dichroism (ECD) calculations, and X-ray diffraction crystallography. A cholesterol 7α-hydroxylase (Cyp7a1) luciferase reporter assay revealed significant anti-cholestatic activities for compounds 1, (+)-4, 6, 7, 12-14, and 16. Additionally, compound 6 demonstrated anti-cholestatic effects through the farnesoid X receptor (FXR)-associated signaling pathways in vitro and in vivo. These findings suggest potential applications for I. Lophanthoides in pharmaceutical development.
Abietanes/pharmacology* ; Molecular Structure ; Animals ; Isodon/chemistry* ; Humans ; Diterpenes/pharmacology* ; Plant Extracts/chemistry*

This study was designed to investigate the effect of silencing microtubule-affinity regulating kinase 4(MARK4)on the apoptosis,inflammatory cytokine release and intestinal barrier protein expression of FHC cells in a lipopolysaccharide(LPS)-induced ulcerative colitis(UC)model,and the underlying molecular mechanisms.Western blot analysis was used to measure the expression levels of MARK4 and apoptosis-related factors including Caspase-1,NLRP3,and GSDMD in colon tissues from both UC patients and healthy individuals,as well as in LPS-induced FHC cell inflammation model.FHC cells was transfected with shRNA to silence MARK4.In control(normal FHC cells),LPS(LPS-stimulated FHC cells),and MARK4-silenced+LPS(shRNA-and LPS-treated FHC cells)groups,the expression levels of Caspase-1,NLRP3,GSDMD,intestinal barrier proteins,and NF-κB pathway-related proteins were assessed by Western blotting.ELISA and RT-qPCR were used to measure the expression levels of inflammatory cytokines IL-1β,IL-6,and TNF-α;flow cytometry was utilized to assess apoptosis.Data showed that both in UC patient colon tissues and the in vitro LPS-induced FHC cell UC inflammation model,there was a significant increase in the expression of MARK4 and apoptosis-related proteins including NLRP3,Caspase-1,and GSDMD.Silencing MARK4 inhibited the expression of these apoptosis-related proteins and downregulated the inflammatory cytokines IL-1β,IL-6,and TNF-α in LPS-induced FHC cells.Silencing MARK4 also reduced apoptosis,increased the expression of intestinal barrier proteins ZO-1,Occludin,and upregulated Claudin2.Gene Set Enrichment Analysis(GSEA)indicated a positive correlation between MARK4 and the NF-κB signaling pathway.Furthermore,silencing of MARK4 inhibited the expression levels of p-P65 and p-IKKα in the NF-κB pathway.In conclusion,MARK4 is significantly upregulated in UC tissues and cells.Silencing MARK4 inhibits the activation of the NF-κB signaling pathway,thereby inhibiting the apoptosis and inflammatory factor expression of UC cells.Thus,MARK4 could be a potential therapeutic target for UC patients.

Objective:This study aims to explore the application value of the "liftoff" modular method in robot-assisted laparoscopic surgery for complex adrenal tumors.Methods:We retrospectively analyzed the clinical data of 15 patients with complex adrenal tumors treated at the General Hospital of Southern Theater Command from May 2022 to June 2023. The cohort comprised 5 males and 10 females with an average age of (47.6±7.8) years and a body mass index (BMI) of 26.5 (23.8-27.9) kg/m 2. Among the patients, 3 had a BMI ≥28 kg/m 2, 2 had diabetes, 6 had hypertension, and 1 had coronary heart disease. Preoperative endocrine hormone examination revealed abnormal blood catecholamines in 5 cases and abnormal blood cortisol in 2 cases. Ultrasound and CT scans indicated that 9 tumors were located on the left side and 6 on the right, with 4 cases showing tumor compression on adjacent large blood vessels or organs. The average tumor diameter was (7.61±2.79) cm, with 10 cases having a diameter ≥ 6 cm. All patients underwent laparoscopic adrenalectomy assisted by robots through the transperitoneal approach. The surgeries were performed in a lateral position under general anesthesia. The "liftoff" modular method was utilized to separate the treatment of adrenal tumors into lateral, medial, dorsal, cephalic, and adrenal renal plane sides. Tumors were appropriately manipulated during the operations to achieve a "liftoff" shape. Different modular dissociation steps were adopted based on the size and location of the left and right adrenal tumors. The left adrenal gland was dissected in the order of medial and dorsal, adrenal renal plane side, and lateral and cephalic sides, while the right adrenal gland was dissected in the order of lateral and dorsal, adrenal renal plane side, and medial and cephalic sides. Postoperative related indicators and follow-up status of patients were recorded and analyzed. Results:All 15 surgeries were successfully completed without any conversions to open adrenalectomy, with an average operation time of 118 (102-130) minutes and an average intraoperative blood loss of 102 (69-163) ml. The postoperative drainage time was 4 (3-5) days, and the postoperative hospital stay was 6 (4-7) days. The postoperative pathological diagnoses included 5 cases of pheochromocytoma, 3 cases of macronodular adrenal hyperplasia, 6 cases of adrenocortical adenoma, and 1 case of myelolipoma. Follow-up for 6-12 months after surgery showed good recovery and no recurrence.Conclusions:The application of the "liftoff" modular method in robot-assisted laparoscopic surgery for complex adrenal tumors is safe and feasible. It efficiently aids in tumor removal and holds significant clinical application value.

AIM: To study the effect of S-methylisothiourea(SMT) on adriamycin(ADM)-induced changes of superoxide dismutase and alutathione peroxidase activities in myocardial tissue of rats.METHODS: Rats were treated with ADM by intraperioneal injection (10.0 mg/kg body weight),and then the ADM-treated rats were intervened by SMT at different dosages by intravenous injection(once a day for three days).The contents of malondialdehyde(MDA) and NO_2~-/ NO_3~-,the activities of manganes superoxied dismutase(MnSOD),copper-zinc superoxide dismutase(Cu-Zn SOD),glutathione peroxidase(GPx) and inducible nitric oxide synthase(iNOS) were determined by spectrophotometry in myocardial tissue.The activity of isoenzymic CK-MB of creatine kinase(CK) was determined by enzymic rate method in serum.The expression of MnSOD mRNA,Cu-ZnSOD mRNA,GPx mRNA and iNOS mRNA were detected by reverse transcription-ploymerase chain reaction.RESULTS: The contents of MDA,NO~-_2/ NO~-_3,and iNOS activity of myocardial tissue,the activity of CK-MB in serum in SMT(5.0,10.0,20.0 mg/kg) intervention groups were significantly lower than those in ADM-treated group(P