N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes

Abstract

Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC 50 )=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 mg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (DCm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltagedependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations X25 mg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 mg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.

Saikosaponin D mitigate pilocarpine‐induced astrocyte injury by regulating the NLRP3/caspase‐1 signaling pathway

“…Although the changes in astrocyte inflammation and oxidative stress can be observed in the epilepsy model, few studies have used PP as an astrocyte inducer in epilepsy models in vitro. Aquino and his colleagues claimed that PP significantly inhibited the viability of astrocytes and up‐regulated intracellular lipid peroxidation (Aquino et al., 2022; Ramazi et al., 2020). According to previous studies, GFAP and C3 are preferentially expressed in lipopolysaccharide (LPS)‐induced A1 reactive astrocytes (Zamanian et al., 2012), while Ptx3 and S100a10 are preferentially expressed in A2 reactive astrocytes (Guo et al., 2020).…”

“…To establish the epilepsy model of astrocytes, we treated astrocytes with gradient PP and found that 1 mM PP induced a significant decrease in the viability of astrocytes, whereas 0.25–0.5 mM PP had no effect on the astrocyte viability. Notably, PP is often used as an inducer in a mouse or rat epilepsy model (Aquino et al., 2022; Garzillo & Mello, 2002; Hubbard et al., 2016; Levesque et al., 2021). Although the changes in astrocyte inflammation and oxidative stress can be observed in the epilepsy model, few studies have used PP as an astrocyte inducer in epilepsy models in vitro.…”

“…In conclusion, mitochondrial dysfunction plays a crucial role in neuronal death induced by seizure activity, and drug therapy based on mitochondrial dysfunction has consistent antiepileptic and neuroprotective effects (Kovac et al., 2012). Previous studies have shown that a variety of substances or drugs can play an anti‐epileptic role by alleviating mitochondrial dysfunction, such as ketones (Kim et al., 2015), exosomes (Xian et al., 2019), N‐Methyl‐(2S, 4R)‐trans‐4‐hydroxy‐L‐proline (Aquino et al., 2022). In our study, we found that SSD treatment significantly improved MMP abnormalities, thereby alleviating PP‐induced mitochondrial dysfunction in astrocytes.…”

Neuroprotective Effects of N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline (NMP) against Amyloid-β-Induced Alzheimer’s Disease Mouse Model

“…The scavenging of reactive oxygen species (O2− and H2O2) by the nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 (NRF2/HO-1) signaling pathway was the primary method to achieve the goal of anti-oxidation, and a promising factor for improving the cognitive impairment seen in AD [40]. Previous reports have shown that NMP exhibits antioxidant behavior towards cytoplasm ROS accumulation and mitochondrial membrane depolarization caused by pilocarpine [41]. In this study, our Western blot analysis revealed that injecting Aβ 1-42 led to a reduction in the expression of NRF2 and HO-1 in the cortexes and hippocampi of mice treated with amyloid beta.…”