Absolute Quantification of Dynamic Cellular Uptake of Small Extracellular Vesicles via Lanthanide Element Labeling and ICP-MS.

Small extracellular vesicles (sEVs) are increasingly reported to play important roles in numerous physiological and pathological processes. Cellular uptake of sEVs is of great significance for functional regulation in recipient cells. Although various sEV quantification, labeling, and tracking methods have been reported, it is still highly challenging to quantify the absolute amount of cellular uptake of sEVs and correlate this information with phenotypic variations in the recipient cell. Therefore, we developed a novel strategy using lanthanide element labeling and inductively coupled plasma-mass spectrometry (ICP-MS) for the absolute and sensitive quantification of sEVs. This strategy utilizes the chelation interaction between Eu3+ and the phosphate groups on the sEV membrane for specific labeling. sEVs internalized by cells can then be quantified by ICP-MS using a previously established linear relationship between the europium content and the particle numbers. High Eu labeling efficiency and stability were demonstrated by various evaluations, and no structural or functional alterations in the sEVs were discovered after Eu labeling. Application of this method revealed that 4020 ± 171 sEV particles/cell were internalized by HeLa cells at 37 °C and 61% uptake inhibition at 4 °C. Further investigation led to the quantitative differential analysis of sEV cellular uptake under the treatment of several chemical endocytosis inhibitors. A 23% strong inhibition indicated that HeLa cells uptake sEVs mainly through the macropinocytosis pathway. This facile labeling and absolute quantification strategy of sEVs with ppb-level high sensitivity is expected to become a potential tool for studying the functions of sEVs in intracellular communication and cargo transportation.

"Multiomics" Analyses Combined with Systems Pharmacology Reveal the Renoprotection of Mangiferin Monosodium Salt in Rats with Diabetic Nephropathy: Focus on Improvements in Renal Ferroptosis, Renal Inflammation, and Podocyte Insulin Resistance.

We explored the protection of mangiferin monosodium salt (MGM) on kidney injury in rats with streptozotocin (STZ)-induced diabetic nephropathy (DN) by "multiomics" analysis combined with systems pharmacology, with a specific focus on ferroptosis, inflammation, and podocyte insulin resistance (IR) signaling events in kidneys. MGM treatment afforded renoprotective effects on rats with STZ-induced DN by alleviating systemic IR-induced renal inflammation and podocyte IR. These mechanisms were correlated mainly with the MGM treatment-induced inhibition of the mitogen-activated protein kinase/nuclear factor-kappa B axis and activation of the phosphorylated insulin receptor substrate 1(Tyr608)/phosphorylated phosphatidylinositol 3-kinase/phosphorylated protein kinase B axis in the kidneys of DN rats. MGM had an ameliorative function in renal ferroptosis in rats with STZ-induced DN by upregulating mevalonate-mediated antioxidant capacities (glutathione peroxidase 4 and ferroptosis suppressor protein 1/coenzyme Q10 axis) and weakening acyl-CoA synthetase long-chain family member 4-mediated proferroptotic generation of lipid drivers in kidneys. MGM may be a promising alternative strategy for the treatment of DN.

Beneficial effects of procyanidin B2 on adriamycin-induced nephrotic syndrome mice: the multi-action mechanism for ameliorating glomerular permselectivity injury.

Despite considerable advances in prevention, diagnosis, and therapy, nephrotic syndrome (NS) remains a significant cause of high morbidity and mortality globally. As a result, there is an urgent need to identify novel effective preventative and therapeutic agents for NS. NS is implicated in glomerular permselectivity injury, which can be attributed to oxidative distress, inflammation, lipid nephrotoxicity, podocyte apoptosis, autophagy dysfunction, and slit diaphragm (SLD) dysfunction. In addition to its well-documented antioxidant potency, procyanidin B2 (PB2) may exhibit pleiotropic effects by targeting various canonical signaling events, such as NF-κB, PPARs, PI3K/Akt, mTOR, and the caspase family. As a result, PB2 may be a promising therapeutic target against NS. To test this hypothesis, we established an Adriamycin (ADR)-induced NS mouse model to evaluate the pleiotropic renoprotective effects of PB2 on NS. Here, we demonstrated that PB2 improves podocyte injury via inhibition of NOX4/ROS and Hsp90/NF-κB to exhibit antioxidant and anti-inflammatory potency, respectively. We also show that PB2 indirectly activates the PI3K/Akt axis by regulating SLD protein levels, resulting in normalized podocyte apoptosis and autophagy function. Further, loss of albumin (ALB) induces lipid nephrotoxicity, which we found to be alleviated by PB2 via activation of PPARα/ß-mediated lipid homeostasis and the cholesterol efflux axis. Interestingly, our results also suggested that PB2 reduces electrolyte abnormalities and edema. In addition, PB2 may contribute protective effects against trace element dys-homeostasis, which, through alleviating serum ALB loss, leads to a protective effect on glomerular permselectivity injury. Taken together, our results reveal that the identified mechanisms of PB2 on NS are multifactorial and involve inhibition of oxidative distress and inflammatory responses, as well as improvements in podocyte apoptosis and autophagy dysfunction, amelioration of lipid nephrotoxicity, and modulation of electrolyte abnormalities and edema. Thus, we provide a theoretical basis for the clinical application of PB2 against NS.

Protective role of selenium in the activities of antioxidant enzymes in piglet splenic lymphocytes exposed to deoxynivalenol.

We evaluated the effects of selenium (Se) on antioxidant enzymes of piglet splenic lymphocytes exposed to deoxynivalenol (DON). We measured cell viability, the activities of several antioxidant enzymes, and lactate dehydrogenase (LDH), as well as total antioxidant capacity (T-AOC) and the levels of malonaldehyde (MDA) and hydrogen peroxide (H2O2). We found that DON exposure increased the concentrations of LDH, MDA, and H2O2 in all experimental groups in a dose-dependent manner, while the concentrations of other antioxidant enzymes were decreased. In Se-pretreated DON-exposed cells, damage to antioxidant enzymes was reduced, especially in the lower-dose DON groups over longer exposure times. These results may indicate that in piglet splenic lymphocytes, Se can alleviate DON-induced damage to antioxidant enzymes by improving glutathione peroxidase activity. Se may function as a potential antioxidative agent to alleviate DON-induced oxidative stress.