Liaoning University of Traditional Chinese Medicine

To explore the N6-methyladenosine (m⁶A) modification mechanism of taurine upregulated gene 1 (TUG1) and whether methyltransferase 3 (METTL3) can promote peroxisome proliferators-activated receptor γ coactivator 1 alpha (PGC-1α) transcription and alleviate mitochondrial dysfunction.

In vitro high glucose (HG)-treated HK-2 cell models and in vivo db/db mice models injected with rAAV-METTL3 via the tail vein were established. The expression levels were determined by RT-qPCR, western blot, and immunohistochemical staining. RNA m⁶A modification was analyzed by the RNase Mazf. The biochemical indicators of mice were detected by enzyme-linked immunosorbent assay. Cell apoptosis was detected by flow cytometry. Histopathological staining was performed to evaluate kidney injury. mtDNA content, mitochondrial complex activity, and ATP were detected by RT-qPCR and detection kits, respectively, per the manufacturer's instructions. Mitochondrial reactive oxygen species production in HK-2 cells incubated with MitoSOX Red and mitochondrial morphology were observed under a fluorescence microscope and transmission electron microscope, respectively. Molecular interactions were verified through RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter gene assay.

METTL3 and TUG1 expression levels decreased in the kidneys of diabetic mice and HG-treated HK-2 cells. Mechanistically, METTL3-mediated m⁶A modification increased the stability of TUG1 in an insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2)-dependent manner. METTL3-mediated m⁶A modification of TUG1 promotes PGC-1α activation, thereby alleviating mitochondrial dysfunction in HG-treated HK-2 cells and db/db mice. Moreover, METTL3 overexpression alleviated kidney injury in db/db mice.

METTL3 targets TUG1/PGC-1α and ameliorates mitochondrial dysfunction in diabetic nephropathy in an IGF2BP2-dependent manner.

Zhishi decoction (ZSD) is one of the most common herb decoctions in traditional Chinese medicine (TCM), and it is used for the treatment of FC. However, its main therapeutic mechanism is not yet clear. This study aims to explore the possible pharmacodynamic material basis and potential molecular mechanism from network pharmacology and molecular docking and verify them through animal experiments.

Firstly, the effective ingredients, potential targets, and key targets of ZSD in the treatment of FC were screened through network pharmacology. Go and KEGG analyses were performed for potential targets. Secondly, molecular docking was used to link the main active components of ZSD with target genes to predict their possible molecular mechanisms. Finally, 30 male BALB/c mice (20±2 g) were randomly divided into five groups (n=6), including the blank group, ZSD groups with two dosages (7.15, 14.3 g/kg), FC model group, and positive group (lactulose group). All the mice were given difenoxate tablets for 14 days to establish FC model except the blank group. Moreover, the mice in the blank group were given the same volume of normal saline. After admination for 14 days, the whole colon tissues were obtained for the analysis of small intestinal propulsion rate, and the expression of P38MAPK in colon tissues of mice was observed via immunohistochemistry and WesterBlot.

In this study, 43 active ingredients in ZSD were identified. Four hundred and thirty potential therapeutic targets were selected, among which AKT1, MAPK12, and MAPK14 were key targets. 164 GO biological processes and 123 KEGG signaling pathways were identified after analysis， such as MAPK signaling pathway, TNF signaling pathway etc. The molecular docking results showed that Prangenin, 4-Hydroxyhomopterocarpin, isoponcimarin, and AKT1, MAPK12, MAPK14 had good binding degree. Additionally, ZSD could relieve the symptoms of FC in mice significantly. Compared with the model group, p38/MAPK positive expression cells and protein expression levels in the colon tissues of ZSD groups significantly increased in a dose-dependent manner (p<0.01).

This study confirmed that ZSD could act on AKT1, MAPK12, and MAPK14 targets to activate the p38/MAPK signaling pathway to relieve FC induced by defenoxate tablets. The further development of ZSD provided a theoretical basis.