Integrative metabolomics and proteomics analyses reveal the mechanism underlying neurotoxicity of rifampicin in HT22 cells.

Stroke is a prevalent and debilitating neurodegenerative condition. Ginsenoside Rg1 has demonstrated neuroprotective properties in the context of stroke. The upregulation of chemokine-like factor 1 (CKLF1) observed in ischemic stroke positions CKLF1 as a promising therapeutic target. However, limited research has explored whether Rg1 can mitigate oxygen-glucose deprivation/reoxygenation (OGD/R)-induced apoptosis in HT22 cells through the modulation of CKLF1. In this study, Na2S2O4 was used to treat HT22 cells to establish the OGD/R model. The effects of different concentrations of Rg1 on cell viability were firstly determined by CCK-8 assay to determine its safe administration range. Subsequently, the level of oxidative stress was assessed by detecting LDH release and antioxidant indexes (CAT, SOD, MDA). Western blotting was used to analyse the expression of CKLF1 and apoptosis-related proteins, and TUNEL staining was used to quantify the apoptosis rate. To explore the cell-cell interactions, a Transwell co-culture system of HT22 and BV-2 cells was established. In this study, the optimal parameters for the OGD/R model were determined: 25mmol/L Na2S2O4 treatment for 2.5h followed by 2.5h of reoxygenation, and a cell inoculation density of 1 × 105 cells/mL for 1 day of culture. Based on the safety assessment, 5, 25, and 50μmol/L Rg1 were selected for intervention. Rg1 significantly decreased LDH release (P ≤ 0.05) and MDA content (P ≤ 0.05) and alleviated oxidative stress. Western blotting showed that Rg1 dose-dependently downregulated the expression of CKLF1 (P ≤ 0.05) and inhibited Caspase-3 and other apoptotic protein activation. In the HT22/BV-2 co-culture system, Rg1 inhibited microglia activation, as shown by reduced NO and IL-1β secretion (P ≤ 0.05). Rg1 attenuates OGD/R injury, reduces oxidative stress and apoptosis in HT22 cells by inhibiting CKLF1 expression and alleviates the inflammatory response in activated BV-2 cells, showing therapeutic potential.

Salidroside intensifies mitochondrial function of CoCl2-damaged HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway

Background and Aims AA Amyloidosis is a multisystemic amyloidosis subtype that develops on the background of various chronic inflammatory etiologies. Urinary omics studies have become a promising tool for elucidating pathophysiology and prognosis of glomerular diseases. However, no urinary omics analysis has been performed focusing on renal AA amyloidosis in literature to the best of our knowledge. Our main aim in this study is to perform a comparative urine proteomic and metabolomic analysis of recently diagnosed renal AA amyloidosis and to investigate the correlation of bioinformatic results with clinical and pathological data. Method Urine samples of 8 recently diagnosed AA amyloidosis (AA), 8 membranous nephropathy (MN) and 6 healthy control group patients were collected before kidney biopsy procedure. Proteomic analyzes were performed with nLC/Q-TOF MS/MS and metabolomic analyzes were performed by GC/MS in all patients. Biopsy specimens were scored according to glomerulosclerosis (G), tubular atrophy (TA) and interstitial fibrosis (IF) grades by two pathologists. Raw spectroscopic data was analyzed using MaxQuant and MS-DIAL programs for proteomic and metabolomic studies, respectively. Statistical analysis of the differences in molecules between study groups were performed with ANOVA and HSD-Tukey tests. Principal component (PCA) and heatmap analyzes were made in R language, while gene ontology (GO), network and functional enrichment analysis of bioinformatic results were performed with PANTHER, STRING and MetaboAnalyst databases. Results In comparison between AA and MN groups, median eGFR values tend to be lower in the AA group (67.6 vs 112 ml/min/1.73 m2 respectively, p = 0.08). Median 24-hour urine protein levels did not show statistically significant difference (9499 vs 9512 mg/day respectively, p = 0.9). Percentage of patients with moderate/severe IF/TA was higher and G score was tend to be in AA group compared to MN group (p values 0.02 and 0.07 for IF/TA and G scores, respectively). As a result of proteomic analysis, a total of 859 proteins were determined. Statistical analysis showed 51 proteins that were significantly differ in AA group compared to the control group. GO and functional enrichment analyzes showed that statistically most significant sub-domains were mainly related with cell-cell adhesion (Figure 1 & 2). In comparative analysis between AA and MN patients, uromodulin (UMOD) was lower in the AA group than in the MN group (log2FC -3.37), whereas ribonuclease 1 (RNASE1) and α-1-microglobulin/bikunin precursor protein (AMBP) were higher in the AA group (log2FC 3.41 and 3.07, respectively). In Spearman correlation analyzes, significant negative correlations were demonstrated between UMOD-proteinuria (r = -0.48, p = 0.03) and between AMBP-eGFR (r = -0.69, p = 0.003) variables. Metabolomic analysis showed 9 metabolites that were significantly different between AA and other study groups. Myo-inositol and urate were higher in AA group compared to MN group, while D-mannitol and N-acetylglutamate were higher in AA group compared to the control group. Significant positive correlation independent of GFR was detected between RNASE1 and urate (r = 0.63, p = 0.01). Conclusion Our study is the first urinary comparative omics analysis performed on renal AA amyloidosis patients to the best of our knowledge. We demonstrated specific protein and metabolites that distinguish AA group from the control and MN groups. Enrichment and GO analyzes between AA and the control group showed a negative enrichment in cell-cell adhesion related sub-domains, suggesting a possible increased urinary shear stress resulting in downregulation of cadherins in AA amyloidosis. In comparative analysis between AA and MN groups, UMOD and AMBP proteins and myo-inositol were thought to be associated with high tubulointerstitial damage, whereas RNASE1 and urate were believed to be related with systemic inflammation and endothelial damage [1].

Activated AMPK-mediated glucose uptake and mitochondrial dysfunction is critically involved in the glutamate-induced oxidative injury in HT22 cell

Curcumin (Cur) induces neuroprotection against brain ischemic injury; however, the mechanism is still obscure. The aim of this study is to explore the potential neuroprotective mechanism of curcumin against oxygen-glucose deprivation/reoxygenation (OGD/R) injury in HT22 cells and investigate whether type-2 superoxide dismutase (SOD2) is involved in the curcumin-induced protection. In the present study, HT22 neuronal cells were treated with 3 h OGD plus 24 h reoxygenation to mimic ischemia/reperfusion injury. Compared with the normal cultured control group, OGD/R treatment reduced cell viability and SOD2 expression, decreased mitochondrial membrane potential (MMP) and mitochondrial complex I activity, damaged cell morphology, and increased lactic dehydrogenase (LDH) release, cell apoptosis, intracellular reactive oxygen species (ROS), and mitochondrial superoxide (P < 0.05). Meanwhile, coadministration of 100 ng/ml curcumin reduced the cell injury and apoptosis, inhibited intracellular ROS and mitochondrial superoxide accumulation, and ameliorated intracellular SOD2, cell morphology, MMP, and mitochondrial complex I activity. Downregulating the SOD2 expression by using siRNA, however, significantly reversed the curcumin-induced cytoprotection (P < 0.05). These findings indicated that curcumin induces protection against OGD/R injury in HT22 cells, and SOD2 protein may mediate the protection.

This study is aimed at exploring the potential of isorhamnetin in protection against diabetes-exacerbated ischemia/reperfusion-induced brain injury and elucidating its action mechanism. After establishment of the model of high glucose (HG)-aggravated oxygen-glucose deprivation and reoxygenation (OGD/R), HT22 cell viability was detected by CCK-8. Lactate dehydrogenase (LDH) activity, casapase-3 activity, and oxidative stress-related markers in HT22 cells were detected by corresponding commercial kits. The apoptosis of HG-treated HT22 cells following OGD/R was observed with TUNEL staining. The level of pro-inflammatory cytokines was examined by ELISA. The expression of Akt/SIRT1/Nrf2/HO-1 signaling-related proteins was assayed by Western blot. The results showed that HG noticeably worsened the OGD/R-induced apoptosis of HT22 cells. Isorhamnetin relieved the HG-aggravated OGD/R-induced apoptosis, inflammatory response, and oxidative stress of HT22 cells. Isorhamnetin alleviated the HG-aggravated OGD/R injury in HT22 cells through Akt/SIRT1/Nrf2/HO-1 signaling pathway. Meanwhile, treatment with Akt inhibitor LY294002 reversed the protective effects of isorhamnetin against HG-aggravated OGD/R injury in HT22 cells. In a conclusion, Isorhamnetin alleviates HG-aggravated OGD/R in HT22 hippocampal neurons through Akt/SIRT1/Nrf2/HO-1 signaling pathway.

Tanshinone IIA inhibited intermittent hypoxia induced neuronal injury through promoting autophagy via AMPK-mTOR signaling pathway

To investigate whether hydrogen-rich water exerts a protective effect against cellular injury by affecting the level of autophagy after oxygen glucose deprivation/reoxygenation (OGD/R) in a mouse hippocampal neuronal cell line (HT22 cells). HT22 cells in logarithmic growth phase were cultured in vitro. Cell viability was detected by cell counting kit-8 (CCK-8) assay to find the optimal concentration of Na2S2O4. HT22 cells were divided into control group (NC group), OGD/R group (sugar-free medium+10 mmol/L Na2S2O4 treated for 90 minutes and then changed to normal medium for 4 hours) and hydrogen-rich water treatment group (HW group, sugar-free medium+10 mmol/L Na2S2O4 treated for 90 minutes and then changed to medium containing hydrogen-rich water for 4 hours). The morphology of HT22 cells was observed by inverted microscopy; cell activity was detected by CCK-8 method; cell ultrastructure was observed by transmission electron microscopy; the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 was detected by immunofluorescence; the protein expression of LC3II/I and Beclin-1, markers of cellular autophagy, was detected by Western blotting. Inverted microscopy showed that compared with the NC group, the OGD/R group had poor cell status, swollen cytosol, visible cell lysis fragments and significantly lower cell activity [(49.1±2.7)% vs. (100.0±9.7)%, P < 0.01]; compared with the OGD/R group, the HW group had improved cell status and remarkably higher cell activity [(63.3±1.8)% vs. (49.1±2.7)%, P < 0.01]. Transmission electron microscopy showed that the neuronal nuclear membrane of cells in the OGD/R group was lysed and a higher number of autophagic lysosomes were visible compared with the NC group; compared with the OGD/R group, the neuronal damage of cells in the HW group was reduced and the number of autophagic lysosomes was notably decreased. The results of immunofluorescence assay showed that the expressions of LC3 and Beclin-1 were outstandingly enhanced in the OGD/R group compared with the NC group, and the expressions of LC3 and Beclin-1 were markedly weakened in the HW group compared with the OGD/R group. Western blotting assay showed that the expressions were prominently higher in both LC3II/I and Beclin-1 in the OGD/R group compared with the NC group (LC3II/I: 1.44±0.05 vs. 0.37±0.03, Beclin-1/β-actin: 1.00±0.02 vs. 0.64±0.01, both P < 0.01); compared with the OGD/R group, the protein expression of both LC3II/I and Beclin-1 in the HW group cells were notably lower (LC3II/I: 0.54±0.02 vs. 1.44±0.05, Beclin-1/β-actin: 0.83±0.07 vs. 1.00±0.02, both P < 0.01). Hydrogen-rich water has a significant protective effect on OGD/R-causing HT22 cell injury, and the mechanism may be related to the inhibition of autophagy.

Dihydromyricetin (DMY) is a natural flavonoid that possesses a wide range of pharmacological properties. The aim of the present study was to determine whether DMY could protect against nerve cell injury following ischemic stroke through antioxidant and neuroprotective effects. The effects of DMY on the viability, oxidative stress and apoptosis of HT22 cells following oxygen-glucose deprivation and re-oxygenation (OGD/R) were examined using MTT, lactate dehydrogenase (LDH), superoxide (SOD), malondialdehyde (MDA), western blot and TUNEL assays. Furthermore, Wnt/β-catenin signaling proteins in OGD/R-stimulated HT22 cells were detected in the presence or absence of DMY. In a separate experiment, the effect of DMY on OGD/R-induced HT22 cell injury was also observed in the presence of the Wnt/β-catenin inhibitor, XAV939. The results demonstrated that DMY had no impact on the survival of untreated HT22 cells, although DMY treatment significantly increased cell viability and inhibited cytotoxicity, oxidative stress and apoptosis following OGD/R. In addition, DMY upregulated the expression of Wnt/β-catenin in OGD/R-stimulated HT22 cells. In conclusion, DMY protected HT22 cells from OGD/R-induced oxidative stress and apoptosis, and its effects may be mediated by the activation of the Wnt/β-catenin signaling pathway.

Emerging evidences suggest that nicotine exerts a neuroprotective effect on Alzheimer's disease (AD), yet the precise mechanism is not fully elucidated. Here, HT22 cells were exposed to amyloid beta protein fragment (Aβ)1-42 to mimic the pathological process of neuron in AD. We hypothesized that cannabinoid receptor CB1 is involved in the nicotine-induced neuroprotection against Aβ1-42 injury in HT22 cells. CB1 expression in HT22 cells was investigated by immunocytochemistry and Western blot. The injury of HT22 cells was evaluated by cellular morphology, cell viability, and lactate dehydrogenase (LDH) release. The apoptosis of HT22 cells was assessed by flow cytometry and expressions of Bcl-2 and Bax. The results demonstrated that nicotine markedly upregulated CB1 expression, increased cell viability, ameliorated cellular morphology, decreased LDH release, and reduced the apoptotic rate of HT22 cells exposed to Aβ1-42 for 24h, while the blockade of CB1 or the inhibition of protein kinase C (PKC) partially reversed the neuroprotection. Furthermore, the blockade of CB1 reversed nicotine-induced PKC activation in HT22 cells exposed to Aβ1-42. These results suggest that CB1 is involved in the nicotine-induced neuroprotection against Aβ1-42 neurotoxicity, and the neuroprotection may be dependent on the activation of PKC.

Autophagy has been implicated in neurodegenerative diseases. Forkhead box O3 (FoxO3) transcription factors promote autophagy in heart and inhibit oxidative damage. Here we investigate the role of FoxO3 transcription factors in regulating autophagy after oxidative stress injury in immortalized mouse hippocampal cell line (HT22). The present study confirms that hydrogen peroxide (H2O2) injury could induce autophagy and FoxO3 activation in HT22 cells. In addition, overexpression of FoxO3 enhanced H2O2-induced autophagy activation and suppressed neuronal cell damage, while knockdown of FoxO3 reduced H2O2-induced autophagy activation and exacerbated neuronal cell injury. Inhibition of autophagy by 3-methyladenine (3-MA) resulted in reduced cell viability, increased production of reactive oxygen species (ROS), promoted nuclear condensation, and decreased expression of antiapoptotic and autophagy-related proteins, indicating that autophagy may have protective effects on H2O2-induced injury in HT22 cells. Moreover, overexpression of FoxO3 prevented exacerbation of brain damage induced by 3-MA. Taken together, these results show that activation of FoxO3 could induce autophagy and inhibit H2O2-induced damage in HT22 cells. Our study demonstrates the critical role of FoxO3 in regulating autophagy in brain.

Oxidative stress is well known to play a pivotal role in hypoxia/reoxygenation (H/R)-induced neuron injury. On the basis of this fact, antioxidative agents have been demonstrated to be neuroprotective. 17-DMAG (HSP90 inhibitor) is reported to have neuroprotective effects in vitro, which may interfere with oxidative stress through reduction in pro-oxidative factors. However, little is known about its effects on H/R-induced neuron injury and the underlying mechanisms. In this study, the effects of 17-DMAG on H/R-treated HT22 cells were investigated. MTT and lactate dehydrogenase (LDH) assays indicated that 17-DMAG led to a dose-dependent recovery of cell viability in H/R-treated HT22 cells. Flow cytometry demonstrated that 17-DMAG inhibited the cell apoptosis induced by H/R in HT22 cells. In addition, Western blot and real-time reverse transcription-polymerase chain reaction indicated that 17-DMAG inhibited the H/R-induced upregulation of Bax/Bcl-2 ratio and cleaved caspase-3 expression. Moreover, our results demonstrated that 17-DMAG promoted the expression of antioxidant enzymes, including manganese superoxide dismutase, catalase, and glutathione peroxidase. As a result, 17-DMAG might resist to H/R-induced oxidative stress. Furthermore, 17-DMAG increased the expression of phosphorylation of Akt (p-Akt) and the heme oxygenase-1 (HO-1), as well as the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in H/R-treated HT22 cells. However, the Akt inhibitor, LY294002, partially hampered the effects of 17-DMAG on the expression of p-Akt, nuclear Nrf2, and HO-1 and cell viability, as well as cell apoptosis induced by H/R in HT22 cells. In conclusion, the findings of our study thus demonstrate that 17-DMAG protects against H/R-induced HT22 cell injury through Akt/Nrf2/HO-1 pathway, which may be associated with its antiapoptotic and antioxidative stress effects.

Objective To explore whether receptor interacting protein (RIP)1/RIP3 pathways participate in glutamate induced cell death in HT-22 neuronal cells and investigate the potential neuroprotection of necrostatin-1 in glutamate induced cell death in HT-22. Methods (1) In vitro cultured mouse hippocampal neuronal HT-22 cells were divided into control group, zVAD group, necrostatin-1 (Nec-1) group, glutamate group, glutamate+zVAD group, glutamate+zVAD+Nec-1 group and glutamate+Nec-1 group; they were treated with zVAD, Nec-1 and glutamate at the final concentrations of 20 μmol/L, 30 μmol/L and 3 mmol/L for 24 h. Cell viability was detected using a luminescence-based commercial kit Cell Titer-Glo (CTG). Necrotic cell death was measured by propidium iodide (PI) and HE stainings. (2) HT-22 cells were divided into control group I, glutamate group I and glutamate+Nec-1 group I; MitoSox Red was used to detect mitochondrial reactive oxygen species (ROS) level. (3) HT-22 cells were divided into control group II, glutamate group II and glutamate+tertiary butyl-hydroxyanisole (BHA) group; the final concentration of BHA was 100 μmol/L; necrotic cell death was measured by PI and HE stainings after 24 h of treatment. (4) HT-22 cells were divided into RIP3 siRNA and control group III, and then, they were transfected with RIP3 siRNA or negative siRNA, respectively; the RIP3 protein expression was determined by Western blotting after 72 h of treatment. (5) HT-22 cells were divided into negative siRNA+Control, RIP3 siRNA, negative siRNA+glutamate and RIP3 siRNA+glutamate groups; the cells were transfected with RIP3 siRNA or Negative siNRA, respectively; 48 h later, the glutamate groups were treated with 3 mmol/L glutamate; PI positive cells and cell viability were measured by PI and HE stainings and CTG at 24 h after glutamate treatment. Results (1) As compared with control group, percentage of PI positive cells was greatly increased and cell viability was decreased in glutamate group and glutamate+zVAD group, with statistically significant differences (P<0.05); as compared with those in the glutamate group, percentage of percentage of PI positive cells was was significantly decreased and cell viability was statistically increased in glutamate+Nec-1 group (P<0.05). (2) ROS level in HT-22 cells of the glutamate group was significantly increased than that in the control group I (P<0.05); however, ROS level in HT-22 cells of glutamate+Nec-1 group I was significantly decreased than that in glutamate group I (P<0.05). (3) Percentage of PI positive cells in the glutamate group II was significantly higher than that in the control group II (P<0.05), and that in the glutamate+BHA group was statistically lower than that in the glutamate group II (P<0.05). (4) The RIP3 protein expression in the RIP3 siRNA group was obviously down-regulated as compared with that in the control group III. (5) As compared with those in the negative siRNA group, percentage of PI positive cells was statistically increased and cell viabilities were statistically decreased in glutamate group (P<0.05); however, percentage of PI positive cells was significantly decreased and cell viability was significantly increased in RIP3 siNRA+glutamate group as compared with those in the glutamate group (P<0.05). Conclusion RIP1/RIP3 pathway and ROS might mediate glutamate induced cell death in HT-22 cells. Key words: Glutamate; Necroptosis; Necrostatin-1; Receptor interacting protein 1/3; HT-22 cell

BackgroundIntracerebral hemorrhage (ICH) is a severe subtype of stroke with high mortality and morbidity. Serpin Family E Member 1 (SERPINE1) has been documented to be upregulated following ICH, however, the participation of SERPINE1 in the development of ICH has never been studied.MethodsHemin was utilized to develop an in vitro model of ICH. Gene levels were evaluated by the use of quantitative reverse transcription polymerase chain reaction, Western blot, as well as enzyme-linked immunoassay assay. The activity of caspase-3 was determined using a commercial kit. Cell viability and apoptosis were assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Terminal deoxynucleotidyl transferase (TdT) d UTP Nick-End Labeling assay.ResultsSERPINE1 was upregulated in hemin-treated HT22 cells. Silencing of SERPINE1 attenuated hemin-induced inhibition of cell viability. Moreover, knockdown of SERPINE1 repressed hemin-induced apoptosis in HT22 cells, as evidenced by the decrease in the number of TUNEL positive cells, caspase-3 activity, and Bax expression, and the increase in Bcl-2 expression. Meanwhile, knockdown of SERPINE1 repressed hemin-induced inflammation in HT22 cells, as indicated by reduced levels of tumor necrosis factor-α, interleukin-6 (IL-6), IL-1β, and inducible nitric oxide synthase. We also found that transforming growth factor-beta 1 (TGF-β1) induced SERPINE1 expression in a dose-dependent manner. Besides, SERPINE1 knockdown attenuated the effects of TGF-β1 on hemin-induced neuronal damage.ConclusionTGF-β1-induced SERPINE1 activation exacerbated hemin-induced apoptosis and inflammation in HT22 cells, manifesting a novel mechanism for ICH progression.

Mitochondrion, as the main energy-supply organelle, is the key target region that determines neuronal survival and death during ischemia. When an ischemic stroke occurs, timely removal of damaged mitochondria is very important for improving mitochondrial function and repairing nerve damage. This study investigated the effect of ligustilide(LIG), an active ingredient of Chinese medicine, on mitochondrial function and mitophagy based on the oxygen and glucose deprivation/reperfusion(OGD/R)-induced injury model in HT22 cells. By OGD/R-induced injury model was induced in vitro, HT22 cells were pre-treated with LIG for 3 h, and the cell viability was detected by the CCK-8 assay. Immunofluorescence and flow cytometry were used to detect indicators related to mitochondrial function, such as mitochondrial membrane potential, calcium overload, and reactive oxygen species(ROS). Western blot was used to detect the expression of dynamin-related protein 1(Drp1, mitochondrial fission protein) and cleaved caspase-3(apoptotic protein). Immunofluorescence was used to observe the co-localization of the translocase of outer mitochondrial membrane 20(TOMM20, mitochondrial marker) and lysosome-associated membrane protein 2(LAMP2, autophagy marker). The results showed that LIG increased the cell viability of HT22 cells as compared with the conditions in the model group. Furthermore, LIG also inhibited the ROS release, calcium overload, and the decrease in mitochondrial membrane potential in HT22 cells after OGD/R-induced injury, facilitated Drp1 expression, and promoted the co-localization of TOMM20 and LAMP2. The findings indicate that LIG can improve the mitochondrial function after OGD/R-induced injury and promote mitophagy. When mitophagy inhibitor mdivi-1 was administered, the expression of apoptotic protein increased, suggesting that the neuroprotective effect of LIG may be related to the promotion of mitophagy.