AIM:This study utilized CRISPR/Cas9 technology to create Retnlb floxp knock-in mice,followed by the application of the Cre-LoxP recombination system to generate intestinal epithelial-specific Retnlb gene knockout mice(Retnlb-CKO).This model was developed to investigate the pathogenic mechanisms of Retnlb in inflammatory bowel disease.METHODS:Female and male C57BL/6N mice,aged 8 weeks with the Retnlbflox/+genotype,were housed togeth-er for breeding.Offsprings were screened to identify those with the Retnlbflox/flox genotype.These mice were then crossed with Vil1-Cre transgenic mice,which express Cre recombinase specifically in intestinal epithelial cells,resulting in Retnlb-flox/+,Cre+mice.Subsequent crosses between Retnlbflox/+,Cre+mice and Retnlbflox/flox mice produced Retnlbflox/flox,Cre+mice(Retnlb-CKO).Six 8-week-old Retnlbflox/flox,Cre+mice and their littermate Retnlbflox/flox mice were selected for experiments.RT-qPCR and immunohistochemistry were used to assess Retnlb mRNA and protein levels in colonic epithelium.Phenotypic observa-tions included body length,weight,diet,and reproductive capability.Tissue-to-body weight ratios were calculated to ana-lyze growth and development.Intestinal barrier integrity and colonic expression of inflammatory factors were evaluated.RESULTS:The conditional gene knockout mouse model with specific deletion of Retnlb in intestinal epithelial cells was successfully established and validated through genetic identification,mRNA and protein analysis.Compared to Retnlbflox/flox mice,Retnlb-CKO mice exhibited no significant differences in body length,weight,diet,or reproductive capability.There were no differences in the ratios of heart,liver,spleen,lung,kidney,and colon weight to body weight,nor were there morphological differences in various tissues.However,the mRNA expression of tight junction proteins ZO-1,Occlu-din,and Claudin3 in colon tissues of Retnlb-CKO mice was significantly reduced(P<0.01).PAS staining and immunohis-tochemistry revealed a significant decrease in the number of goblet cells and lysozyme-positive cells in the colon tissues of Retnlb-CKO mice(P<0.01).HE staining showed no obvious pathological change in colon tissues of Retnlb-CKO mice.RT-qPCR further demonstrated a significant downregulation of pro-inflammatory factors NLRP3,interleukin-6(IL-6),IL-1β,and tumor necrosis factor-α(TNF-α)in colon tissues(P<0.01),along with significant downregulation of inflamma-tion signaling pathway proteins TLR4,MyD88,and NF-κB(P<0.01).CONCLUSION:A conditional colon epithelial cell Retnlb gene knockout mouse model was successfully constructed and validated.The absence of Retnlb in colon cells led to impaired intestinal barrier function,decreased mRNA expression of pro-inflammatory factors in colon tissue,and downregulation of mRNA expression of inflammatory pathway proteins TLR4,MyD88,and NF-κB.

Intrahepatic cholestasis of pregnancy (ICP), as an idiopathic disease, often occurs in the second and third trimester of pregnancy. It is characterized by maternal pruritus, abnormal liver function and raised serum bile acids which are resolved rapidly and spontaneously after delivery. It has been reported that abnormal intestinal microbiota and endogenous bile acids play important roles in the progression of cholestatic liver diseases. Dramatic changes in metabolic hormone levels and energy metabolism during pregnancy disrupt the microbiota composition and bile acid pool as well. Altered intestinal permeability, overaccumulation of bile acids, aberrant expression of bile acid transporters and nuclear receptors are all involved in the pathogenesis of cholestasis. This paper reviews the changes of intestinal flora and the mechanism of gastrointestinal microbiota in intrahepatic cholestasis of pregnancy, explore the potential treatments for ICP.

Intrahepatic cholestasis of pregnancy (ICP), as an idiopathic disease, often occurs in the second and third trimester of pregnancy. It is characterized by maternal pruritus, abnormal liver function and raised serum bile acids which are resolved rapidly and spontaneously after delivery. It has been reported that abnormal intestinal microbiota and endogenous bile acids play important roles in the progression of cholestatic liver diseases. Dramatic changes in metabolic hormone levels and energy metabolism during pregnancy disrupt the microbiota composition and bile acid pool as well. Altered intestinal permeability, overaccumulation of bile acids, aberrant expression of bile acid transporters and nuclear receptors are all involved in the pathogenesis of cholestasis. This paper reviews the changes of intestinal flora and the mechanism of gastrointestinal microbiota in intrahepatic cholestasis of pregnancy, explore the potential treatments for ICP.

Objective:To establish the reference values of stimulation single fiber electromyography (SFEMG) in orbicularis oculi, and to explore its sensitivity in repetitive nerve stimulation (RNS) negative ocular myasthenia gravis (OMG) patients, and the relationship between jitter and various clinical parameters.Methods:Thirty-two healthy volunteers were included to establish the reference value of normal controls from January 2019 to December 2019. From December 2019 to January 2023, 36 OMG patients with negative RNS were collected. Quantitative MG score (QMGS) was performed, neostigmine test and antibody titers as well as thymus CT results were recorded. One side of the orbicularis oculi muscle was tested with a disposable concentric needle electrode in stimulation SFEMG, and the mean consecutive difference (MCD) value was calculated, which was compared with the average MCD value and upper limit of individual values in normal controls to evaluate whether the jitter was abnormal. Spearman correlation analysis of abnormal mean MCD values with QMGS and antibody titer was conducted.Results:Among the 32 healthy volunteers, there were 13 males and 19 females, the age was (46.8 ±18.7) years, and the MCD was (19.0 ±4.4) μs. The upper limit of the reference value was 27.7 μs for average MCD, and 37.4 μs for 10% individual values. Among 36 OMG patients negative at RNS tests, 20 were male and 16 were female, with a age of (37.2 ±17.0) years. The MCD was (29.9 ±14.7) μs, and Jitter was abnormal in 29 patients (81%). Among them, 20 (20/25) patients were antibody positive, 6 (6/26) patients had thymic hyperplasia, and 7 (7/26) patients had thymoma. The QMGS was 3(2, 4). There were 7 patients (19%) with normal jitter, whose QMGS was 3(2, 4). Among the patients with normal Jitter, 5 (5/5) patients were antibody positive, 2 (2/6) patients had thymic hyperplasia. There was no statistically significant difference in clinical indicators between the two groups of patients with abnormal or normal jitter. There was no significant correlation in antibody titer or QMGS with abnormal mean MCD value. Conclusions:The upper limit of the mean MCD value in the normal controls is 27.7 μs. The upper limit of a single value is 37.4 μs. Its sensitivity for OMG patients with RNS negative is 81%, and the abnormal mean MCD value does not show a significant correlation with various clinical indicators. Abnormal jitter indicates dysfunction of neuromuscular junction transmission, which is an important neuroelectrophysiological indicator for MG patients and is suitable for RNS negative patients. Orbicularis oculi muscle stimulation SFEMG provides a reliable and sensitive electrophysiological means for functional evaluation of neuromuscular junction.