Fan Yuzhuo,Li Jiarui,Huang Lvzhen,Wang Kai,Zhao Mingwei
Introduction: The goal of this study was to investigate the presence of ADORA2A-DRD2 heterodimers in human retinal pigment epithelial (RPE) cells; determine if 7-methylxanthine (7-MX), a nonselective adenosine receptor antagonist which was used to control myopia progression, can influence the behavior of RPE cells through the ADORA2A-DRD2 receptor pathway; and assess the changes in the expression of signaling molecules during cellular signal transduction. Methods: Human RPE cells were cultured in vitro in the presence or absence of 7-MX. Cell proliferation was evaluated with the CCK-8 assay. Apoptosis and necrosis rates were determined by annexin V–FITC/propidium iodide staining and flow cytometry. Immunofluorescence and coimmunoprecipitation were used to examine the protein expression and colocalization of ADORA2A and DRD2 in RPE cells. ADORA2A and DRD2 were knocked down with small interfering RNAs (siRNAs). Changes in the protein expression of ERK1/2 and phospho-ERK1/2 (pERK 1/2), which are signaling molecules downstream of dopamine receptors, were evaluated by Western blot analysis. Results: Immunofluorescence and coimmunoprecipitation showed that ADORA2A and DRD2 were colocalized in RPE cells. The expression of ADORA2A in RPE cells was inhibited by treatment with 50 µmol/L 7-MX for 48 h, and the expression of DRD2, ERK1/2, and pERK1/2 was increased after treatment with 50 µmol/L 7-MX for 48 h. After siRNA-mediated knockdown of DRD2 in RPE cells and further treatment with 50 µmol/L 7-MX for 48 h, the expression of DRD2 was nearly restored to the level observed in the native control. At the experimental concentrations, 7-MX and siRNAs did not affect the proliferation or apoptosis of human RPE cells. Conclusions: ADORA2A and DRD2 heterodimers were present in RPE cells. 7-MX may affect the behaviors of RPE cells through the ADORA2A-DRD2 receptor pathway. 7-MX is an inhibitor of ADORA2A receptors that can prevent inhibition of the DRD2 receptor pathway and increase DRD2 receptor pathway activity. This phenomenon may explain the mechanism of action through which 7-MX can control myopia progression
Hepatitis B virus (HBV) can be completely suppressed after antiviral treatment; however, some patients with chronic hepatitis B (CHB) exhibit elevated alanine aminotransferase (ALT) levels and sustained disease progression. This study provides novel insights into the mechanism and potential predictive biomarkers of persistently elevated ALT (PeALT) in patients with CHB after complete viral inhibition. Patients having CHB with undetectable HBV DNA at least 12 months after antiviral treatment were enrolled from a prospective, observational cohort. Patients with PeALT and persistently normal ALT (PnALT) were matched 1:1 using propensity score matching. Correlations between plasma metabolites and the risk of elevated ALT were examined using multivariate logistic regression. A mouse model of carbon tetrachloride-induced liver injury was established to validate the effect of key differential metabolites on liver injury. Of the 1238 patients with CHB who achieved complete viral suppression, 40 (3.23%) had PeALT levels during follow-up (median follow-up: 2.42 years). Additionally, 40 patients with PnALT levels were matched as controls. Ser-Phe-Ala, Lys-Ala-Leu-Glu, 3-methylhippuric acid, 3-methylxanthine, and 7-methylxanthine were identified as critical differential metabolites between the two groups and independently associated with PeALT risk. Ser-Phe-Ala and Lys-Ala-Leu-Glu levels could be used to discriminate patients with PeALT from those with PnALT. Furthermore, N -acetyl- l -methionine (NALM) demonstrated the strongest negative correlation with ALT levels. NALM supplementation alleviated liver injury and hepatic necrosis induced by carbon tetrachloride in mice. Changes in circulating metabolites may contribute to PeALT levels in patients with CHB who have achieved complete viral suppression after antiviral treatment.
Chang Liu,Yinuo Wu,Huizhe Zhao,Xiangyu Gu,Jinyang Gu,Mengmeng Zhao,Shangci Zuo,Pengchao Wang
7-Methylxanthine (7-MX), a caffeine (1,3,7-trimethylxanthine) derivative, has gained significant attention as a potential drug for myopia treatment. However, the efficient production of this valuable compound poses challenges: Isolation and chemical synthesis of 7-MX are both difficult to realize due to their poor yields and high costs. Therefore, developing efficient biosynthetic pathways has emerged as a promising alternative strategy. This study aimed to establish an efficient, low-cost, and pollutant-free biosynthetic process for producing 7-MX from caffeine, in which the biosynthetic process is achieved by utilization of ndmA, ndmB, and modified ndmD genes. Moreover, the rich caffeine in coffee waste can be used as an ideal substrate for this reaction, which can reduce the production cost and treat the caffeine in coffee waste residue to promote secondary utilization. By optimizing the gene expression, constructing cofactor regeneration system composed of frmA, frmB, and FDH to regenerate NADH to remove the bottleneck, and engineering Escherichia coli for high-density fermentation, we increased the production of 7-MX to an unprecedented 8.37 g/L. This approach represents the most efficient method thus far for producing 7-MX from caffeine and provides insights into synthesizing other valuable methylxanthines.
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