The contribution of a novel PHEX gene mutation to X-linked hypophosphatemic rickets: a case report and an analysis of the gene mutation dosage effect in a rat model.

A Chinese family was identified to have two patients with rickets, an adult female and a male child (proband), both exhibiting signs related to X-linked hypophosphatemic rickets (XLH). Gene sequencing analysis revealed a deletion of adenine at position 1985 (c.1985delA) in the PHEX-encoding gene. To investigate the relationship between this mutation and the pathogenicity of XLH, as well as analyze the effects of different dosages of PHEX gene mutations on clinical phenotypes, we developed a rat model carrying the PHEX deletion mutation. The CRISPR/Cas9 gene editing technology was employed to construct the rat model with the PHEX gene mutation (c.1985delA). Through reproductive procedures, five genotypes of rats were obtained: female wild type (X/X), female heterozygous (-/X), female homozygous wild type (-/-), male wild type (X/Y), and male hemizygous (-/Y). The rats with different genotypes underwent analysis of growth, serum biochemical parameters, and bone microstructure. The results demonstrated the successful generation of a stable rat model inheriting the PHEX gene mutation. Compared to the wild-type rats, the mutant rats displayed delayed growth, shorter femurs, and significantly reduced bone mass. Among the female rats, the homozygous individuals exhibited the smallest body size, decreased bone mass, shortest femur length, and severe deformities. Moreover, the mutant rats showed significantly lower blood phosphorus concentration, elevated levels of FGF23 and alkaline phosphatase, and increased expression of phosphorus regulators. In conclusion, the XLH rat model with the PHEX gene mutation dosage demonstrated its impact on growth and development, serum biochemical parameters, and femoral morphology.

Integrating pharmacokinetics and network analysis to investigate the mechanism of Moutan Cortex in blood-heat and blood stasis syndrome.

BACKGROUND: Raw Moutan Cortex (RMC) has been used in China and other Asian countries for thousands of years. Its medical application is the treatment of cooling blood and promoting blood circulation. However, its therapeutic mechanism is still undefined. METHODS: In this study, the pharmacokinetics strategy that integrated network analysis was employed to explore the mechanism of RMC in blood-heat and blood stasis syndrome (BHS) model rats. Firstly, Ultra-High performance Liquid Chromatography coupled with Diode Array Detector (UHPLC-DAD) method was developed to determine nine absorbed compounds in rat serum in BHS and normal rats after oral administration of RMC extract respectively. Then the pharmacology network was established based on the relationship between nine compounds absorbed into the blood and BHS targets. Finally, the predicted hub targets were validated experimentally in human umbilical vein endothelial cells (HUVECs). RESULTS: Pharmacokinetic study showed that the pharmacokinetic parameters of nine absorbed compounds had significant differences between BHS and normal groups (p < 0.05). Network analysis showed that 8 target genes, namely, F2, F10, F7, PLAU, MAPK14, MAPK10, AKT1, and NOS3 may be the primary targets regulated by RMC for the treatment of BHS. Among them, targets (F2, F10, F7 and MAPK14, MAPK10, AKT) and 4 active ingredients (paeonol, paeoniflorin, quercetin and oxypaeoniflorin) were selected for evaluating the reliability in vitro experiments, which revealed that the mechanism of RMC against BHS syndrome may inhibit inflammatory pathways and regulate coagulation cascades pathway for cooling and promoting blood circulation. CONCLUSION: The proposed pharmacokinetics study integrated network analysis strategy provides a combination method to explore the therapeutic mechanism of RMC on BHS.

Nine absorbed components pharmacokinetic of raw and processed Moutan Cortex in normal and blood-heat and hemorrhage syndrome model rats.

Raw Moutan Cortex (RMC) and Processed Moutan Cortex (PMC) have a long history of use in China and other Asian countries. In this study, a rapid and accurate ultra-high-pressure liquid chromatography coupled with diode array detector (UHPLC-DAD) method was developed and validated for the simultaneous determination of nine absorbed compounds of RMC/PMC. After extraction by protein precipitation with methanol from plasma, the analytes were separated on an Acquity UPLC® BEH Shield RP18 column (2.1 × 100 mm, 1.7 µm, Waters, USA). Acetonitrile (A) and 0.1% (v/v) formic acid in water (B) were selected as the mobile phase to perform gradient elution. The linearity of nine analytes was >0.9915. The intra- and inter-assay precision (RSD) values were within 11.18%, and accuracy ranged from 91.32 to 101.29%. Suitable stability, matrix effect and extraction recoveries were also obtained. The validated method was applied to compare the pharmacokinetics of RMC and PMC in Blood-Heat and Hemorrhage Syndrome Model and normal rats. The results revealed that processing and the pathological state could influence the pharmacokinetic characteristics of compounds in RMC/PMC. The study willbe useful for further studies on pharmacokinetics and clinical application of raw and processed Moutan Cortex.