SuperKine™ West Pico PLUS Chemiluminescent Substrate

Glucose metabolites were detected with mass spectrometry. The mRNA levels of glucose transporters (GLUTs), metabolic enzymes, and inflammatory mediators were detected by quantitative RT-PCR. The protein expression of GLUTs was studied by immunofluorescence staining, Western blotting, and flow cytometry. Glucose uptake was measured by using fluorescent glucose analog. Human nasal epithelial cells (HNECs) were cultured. Bioenergetic analysis was performed with Seahorse XF analyzer. Gene expression in HNECs was profiled by RNA sequencing.

Dyslipidemia and inflammation have great roles in the development of diabetic nephropathy (DN). Oleanolic acid (OA) is a natural triterpenoid that possesses multiple pharmacological properties including anti-oxidation, anti-inflammatory and hypoglycemia. In the present study, the effects of OA on diabetic kidney disease (DKD) and its underlying mechanisms were investigated in DKD rats. Twenty-five of a total thirty-five male Sprague-Dawley (SD) rats were used to establish for Type 2 diabetes mellitus (T2DM) model by high-fat diet combined with streptozotocin (STZ). Then rats were randomly assigned into four group: control group (n = 10), T2DM group (n = 9), OA (50 mg/kg) group (n = 7), OA (100 mg/kg) group (n = 8). Rats were sacrificed at the end of 18 weeks after feeding by intraperitoneal injection of pentobarbital sodium. Body weight (BW), fasting blood glucose (FBG), kidney weight (KW), serum lipid, 24-h urinary microalbumin (UMA), serum creatinine (Scr) and uric acid (UA) were measured. Histopathological changes were observed by PAS staining and electron microscope. The expressions of nephrin, CD68, Collagen-IV, AMPK, p-AMPK, PGC-1Alpha, TLR4, NF-kB and TGF-Beta1 in kidney were also detected by immunohistochemistry or western blot. OA significantly decreased the levels of FBG, kidney index (KI), serum lipid levels, 24 h UMA, Scr, UA in diabetic rats. Additionally, OA obviously attenuated renal lipid accumulation and renal structure abnormalities in diabetic rats. Furthermore, the expression levels of nephrin, p-AMPK/AMPK, PGC-1Alpha were elevated, while CD68, Collagen-IV, TLR4, NF-kB and TGF-Beta1 expressions were decreased in renal tissues of OA treated diabetic rats. OA showed dose-independent. OA can alleviate renal injury in diabetic rats through improving lipid metabolism and inflammation via AMPK/PGC-1α and TLR4/NF-κB signaling pathway.

Perturbations in transforming growth factor- Beta (TGF-Beta) signaling can lead to a plethora of diseases, including cancer. Mutations and posttranslational modifications (PTMs) of the partner of Smad complexes contribute to the dysregulation of TGF-Beta signaling. Here, we reported a PTM of Smad4, R361 methylation, that was critical for Smad complexes formation and TGF-Beta signaling activation. Through mass spectrometric, co-immunoprecipitation (Co-IP) and immunofluorescent (IF) assays, we found that oncogene protein arginine methyltransferase 5 (PRMT5) interacted with Smad4 under TGF-Beta1 treatment. Mechanically, PRMT5 triggered Smad4 methylation at R361 and induced Smad complexes formation and nuclear import. Furthermore, we emphasized that PRMT5 interacting and methylating Smad4 was required for TGF-Beta1-induced epithelial-mesenchymal transition (EMT) and colorectal cancer (CRC) metastasis, and Smad4 R361 mutation diminished PRMT5 and TGF-Beta1-induced metastasis. In addition, highly expressed PRMT5 or high level of Smad4 R361 methylation indicated worse outcomes in clinical specimens analysis. Collectively, our study highlights the critical interaction of PRMT5 and Smad4 and the roles of Smad4 R361 methylation for controlling TGF-Beta signaling during metastasis. We provided a new insight for Smad4 activation. And this study indicated that blocking PRMT5-Smad4 signaling might be an effective targeting strategy in Smad4 wide type CRC.