Objective:To investigate the changes and to establish a reference interval of bile acid profile of healthy pregnant women in the second and third trimesters of pregnancy in our hospital.Methods:A total of 298 healthy singleton pregnant women who underwent prenatal examination in the Department of Obstetrics of Zhejiang Provincial People′s Hospital from July 2019 to August 2020 were enrolled in this study. The overnight fasting serum samples were collected from all subjects during their second and third trimesters of pregnancy. The concentrations of 15 bile acids(cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid and their glycine-and taurine-conjugated types)were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The characteristics of changes were analyzed and the reference intervals were determined for the second and third trimesters. The concentrations of 15 bile acids and total bile acids were skewed-distributed, and 99 percentiles (P 99) were used to represent the unilateral upper limit of the reference interval. Results:There was significant difference in the serum levels of glycine cholic acid (GCA), taurocholic acid (TCA), glycine ursodeoxycholic acid (GUDCA) and lithocholic acid (LCA) between the second and third trimesters healthy pregnant females ( P<0.05). For other 11 bile acids, there was no significant difference. The levels of total bile acids, primary or secondary bile acids, free or conjugated bile acids (glycine-bound and taurine-bound bile acids) were stable with gestation. Conclusion:Primary, secondary or free, and conjugated bile acids in healthy pregnant women remained stable at T 2 and T 3, with significant differences in only a few subtypes of bile acids. While the correlation between glycine-bound and taurine-bound bile acids showed a weakening trend at T 3 ( P<0.05). It is necessary to establish reference intervals of bile acids for healthy pregnant women in this area. This study provided data support for future research on related diseases during pregnancy.

ObjectiveThis study aimed to investigate the intervention effect of Renqing Mangjue on the malignant transformation of gastric mucosal epithelial cells induced by N-methyl-N′-nitro-N-nitrosoguanidine （MNNG） and to explore its molecular mechanism in preventing precancerous lesions of gastric cancer based on the cyclic guanosine monophosphate （cGMP）/protein kinase G （PKG）/mitogen-activated protein kinase （MEK）/extracellular signal-regulated kinase （ERK） signaling pathway. MethodsHuman gastric mucosal epithelial cells （GES-1） were initially induced by MNNG to establish a precancerous cell model （MC cells）. The effective concentration of MNNG for inducing malignant transformation in GES-1 cells was screened using the cell proliferation activity decection （CCK-8） assay， and the effective concentration of Renqing Mangjue for inhibiting the proliferation of transformed GES-1 cells was also determined. GES-1 cells were divided into a blank control group， a model group， and treatment groups with Renqing Mangjue at concentrations of 1， 3， 10， and 30 mg·L^-1. Furthermore， the effects of Renqing Mangjue on the migratory ability and epithelial-mesenchymal transition （EMT） characteristics of GES-1 malignant transformed cells were evaluated using Transwell migration assays， wound healing assays， and real-time quantitative reverse transcription polymerase chain reaction （Real-time PCR）. Additionally， candidate chemical components and target sites of Renqing Mangjue were obtained from the TCMIP v2.0 database， and disease targets at various stages of gastric cancer precursors were sourced from the Gene Expression Omnibus （GEO） database. Pathway enrichment analysis was performed using the Metascape database to predict the potential mechanisms of action of Renqing Mangjue. Finally， the protective mechanism of Renqing Mangjue against gastric cancer precursors was validated through Western blot analysis. ResultsAt a concentration of 20 μmol·L^-1， MNNG exhibited an inhibition rate of approximately 50% on GES-1 cells （P<0.01）， and at this concentration， the GES-1 cells displayed biological characteristics indicative of malignant transformation. In contrast， Renqing Mangjue had no significant effect on the proliferation of normal GES-1 cells， but significantly inhibited the proliferation of MC cells （P<0.01） and markedly reduced their migratory capacity （P<0.01）. Moreover， it also increased the mRNA expression level of E-cadherin during the EMT process （P<0.05）， while inhibiting the expression of both N-cadherin and the transcription factor Snail mRNA （P<0.05， P<0.01）. Network predictions suggested that Renqing Mangjue may prevent gastric cancer precursors through modulating the cGMP/PKG and MAPK/ERK signaling pathways. Furthermore， Western blot results indicated that Renqing Mangjue upregulated the expression of PKG and NPRB （B-type natriuretic peptide receptor） proteins in the cGMP/PKG pathway （P<0.01）， while downregulating the expression of the downstream proteins MEK and ERK （P<0.05， P<0.01）. ConclusionIn summary， Renqing Mangjue can prevent gastric cancer precursors by inhibiting the proliferation and migration of malignant transformed GES-1 cells， thereby delaying the EMT process. The underlying mechanisms may be related to the activation of the cGMP/PKG pathway and the inhibition of the MEK/ERK signaling pathway.