FTO (Fat-Mass and Obesity-Associated Protein) Participates in Hemorrhage-Induced Thalamic Pain by Stabilizing Toll-Like Receptor 4 Expression in Thalamic Neurons

Background and Purpose: Hemorrhage-caused gene changes in the thalamus likely contribute to thalamic pain genesis. RNA N 6 -methyladenosine modification is an additional layer of gene regulation. Whether FTO (fat-mass and obesity-associated protein), an N 6 -methyladenosine demethylase, participates in hemorrhage-induced thalamic pain is unknown. Methods: Expression of Fto mRNA and protein was assessed in mouse thalamus after hemorrhage caused by microinjection of Coll IV (type IV collagenase) into unilateral thalamus. Effect of intraperitoneal administration of meclofenamic acid (a FTO inhibitor) or microinjection of adeno-associated virus 5 (AAV5) expressing Cre into the thalamus of Fto fl/fl mice on the Coll IV microinjection–induced TLR4 (Toll-like receptor 4) upregulation and nociceptive hypersensitivity was examined. Effect of thalamic microinjection of AAV5 expressing Fto (AAV5- Fto ) on basal thalamic TLR4 expression and nociceptive thresholds was also analyzed. Additionally, level of N 6 -methyladenosine in Tlr4 mRNA and its binding to FTO or YTHDF2 (YTH N 6 -methyladenosine RNA binding protein 2) were observed. Results: FTO was detected in neuronal nuclei of thalamus. Level of FTO protein, but not mRNA, was time-dependently increased in the ipsilateral thalamus on days 1 to 14 after Coll IV microinjection. Intraperitoneal injection of meclofenamic acid or adeno-associated virus-5 expressing Cre microinjection into Fto fl/fl mouse thalamus attenuated the Coll IV microinjection–induced TLR4 upregulation and tissue damage in the ipsilateral thalamus and development and maintenance of nociceptive hypersensitivities on the contralateral side. Thalamic microinjection of AAV5- Fto increased TLR4 expression and elicited hypersensitivities to mechanical, heat and cold stimuli. Mechanistically, Coll IV microinjection produced an increase in FTO binding to Tlr4 mRNA, an FTO-dependent loss of N 6 -methyladenosine sites in Tlr4 mRNA and a reduction in the binding of YTHDF2 to Tlr4 mRNA in the ipsilateral thalamus. Conclusions: Our findings suggest that FTO participates in hemorrhage-induced thalamic pain by stabilizing TLR4 upregulation in thalamic neurons. FTO may be a potential target for the treatment of this disorder.

Analysis of the mechanism of Aldo-keto reductase dependent cis-platin resistance in HepG2 based on transcriptomic and NADH metabolic analysis

Aldo-keto oxidoreductase (AKR) inhibitors could reverse several cancer cells' resistance to Cis-platin, but their role in resistance remains unclear. Our RNA-seq results showed de novo NAD biosynthesis-related genes, and NAD(P)H-dependent oxidoreductases were significantly upregulated in Cis-platin-resistant HepG2 hepatic cancer cells (HepG2-RC cells) compared with HepG2 cells. Knockdown of AKR1Cs could increase Cis-platin sensitivity in HepG2-RC cells about two-fold. Interestingly, the AKR1C inhibitor meclofenamic acid could increase Cis-platin sensitivity of HepG2-RC cells about eight-fold, indicating that knockdown of AKR1Cs only partially reversed the resistance. Meanwhile, the amount of total NAD and the ratio of NADH/NAD+ were increased in HepG2-RC cells compared with HepG2 cells. The increased NADH could be explained as a directly operating antioxidant to scavenge radicals induced by Cis-platin. We report here that NADH, which is produced by NAD(P)H-dependent oxidoreductases, plays a key role in the AKR-associated Cis-platin resistance of HepG2 hepatic cancer cells.

The degradation efficiency and mechanism of meclofenamic acid in aqueous solution by UV irradiation

The photodegradation of meclofenamic acid (MCFA) was investigated using UV irradiation process. Parameters affecting the photodegradation process such as dissolved oxygen, reaction temperature, solution pH, initial MCFA concentration, humic acid, cations and anions were carried out. The intermediate products were identified by HPLC-MS analysis, and a tentative photodegradation pathway of MCFA was proposed. Results show that the photodegradation of MCFA can be modeled by the first-order kinetics. The photodegradation efficiency increases with the decreased concentration of humic acid or the decreased initial MCFA concentration, while the reaction temperature and the inorganic ions have no significant effect on the MCFA removal. It has found that the optimal initial solution pH for MCFA degradation is about 7.0. Finally, the HPLC-MS results reveal that the Cl atom in the MCFA molecule would form Cl