The function of MDM2-OE on H/R-induced H9c2 cells was hindered by STEAP3 overexpression.

A-B. Western blot was used to detect IL-1β, IL-6 and TNF-α expression in H/R-induced H9c2 cells after Xinnaotongluo liquid and/or MDM2-OE treatment. C-D. Western blot was used to detect Bax, Bcl-2 and cleaved-caspase 3 expression in H/R-induced H9c2 cells after Xinnaotongluo liquid and/or MDM2-OE treatment. E-F. Western blot was used to detect GPX4, MDM2 and SLC7A11 expression in H/R-induced H9c2 cells after Xinnaotongluo liquid and/or MDM2-OE treatment. G. The iron level of H/R-induced H9c2 cells was measured after Xinnaotongluo liquid and/or MDM2-OE treatment. **p<0.01 vs. control, ##p<0.01 vs. H/R+Xinnaotongluo liquid, &&p<0.01 vs. H/R+MDM2-OE+Xinnaotongluo liquid.</p

MDM2 was downregulated in H/R-induced H9c2 cells, and MDM2 overexpression helped Xinnaotongluo liquid to alleviate oxidative damage of H/R-induced H9c2 cells.

A-B. Western blot was used to detect MDM2 expression in H/R-induced H9c2 cells after Xinnaotongluo liquid treatment. C-D. Western blot was used to detect MDM2 expression in H9c2 cells after MDM2-OE transfection. E. CCK-8 assay was used to detect the viability of H9c2 cells after Xinnaotongluo liquid and/or MDM2-OE treatment. F-I. The levels of GSH, SOD, MDA, and LDH were detected by corresponding commercial assay kits after Xinnaotongluo liquid and/or MDM2-OE treatment. **p<0.01 vs. control, ##p<0.01 vs. H/R, &&p<0.01 vs. H/R+Xinnaotongluo liquid, @@p<0.01 vs. H/R+MDM2-OE.</p

MDM2 overexpression contributed to the function of Xinnaotongluo liquid on the inflammation, apoptosis and ferroptosis of H/R-induced H9c2 cells.

A-B. Western blot was used to detect IL-1β, IL-6 and TNF-α expression in H/R-induced H9c2 cells after Xinnaotongluo liquid and/or MDM2-OE treatment. C-D. Western blot was used to detect Bax, Bcl-2 and cleaved-caspase 3 expression in H/R-induced H9c2 cells after Xinnaotongluo liquid and/or MDM2-OE treatment. E-F. Western blot was used to detect GPX4, MDM2 and SLC7A11 expression in H/R-induced H9c2 cells after Xinnaotongluo liquid and/or MDM2-OE treatment. G. The iron level of H/R-induced H9c2 cells was measured after Xinnaotongluo liquid and/or MDM2-OE treatment. **p<0.01 vs. control, ##p<0.01 vs. H/R, &&p<0.01 vs. H/R+Xinnaotongluo liquid, @@p<0.01 vs. H/R+MDM2-OE.</p

STEAP3 overexpression reversed the function of MDM2-OE on H/R-induced H9c2 cells.

A-B. Western blot was used to detect MDM2, p53 and STEAP3 expression in H9c2 cells after MDM2-OE transfection. C. CCK-8 assay was used to detect the viability of H9c2 cells after Xinnaotongluo liquid, MDM2-OE and STEAP3-OE treatment. D-G. The levels of GSH, SOD, MDA, and LDH were detected by corresponding commercial assay kits after Xinnaotongluo liquid, MDM2-OE and STEAP3-OE treatment. **p<0.01 vs. control, ##p<0.01 vs. H/R+Xinnaotongluo liquid, &&p<0.01 vs. H/R+MDM2-OE+Xinnaotongluo liquid.</p

Development of a Method and Validation for the Quantitation of FruArg in Mice Plasma and Brain Tissue Using UPLC–MS/MS

Aged garlic extract (AGE) is a popular nutritional supplement and is believed to promote health benefits by exhibiting antioxidant and anti-inflammatory activities and hypolipidemic and antiplatelet effects. We have previously identified <i>N</i>-α-(1-deoxy-d-fructos-1-yl)-l-arginine (FruArg) as a major contributor to the bioactivity of AGE in BV-2 microglial cells whereby it exerted a significant ability to attenuate lipopolysaccharide-induced neuroinflammatory responses and to regulate the Nrf2-mediated antioxidant response. Here, we report on a sensitive ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) protocol that was validated for the quantitation of FruArg in mouse plasma and brain tissue samples. Solid-phase extraction was used to separate FruArg from proteins and phospholipids present in the biological fluids. Results indicated that FruArg was readily absorbed into the blood circulation of mice after intraperitoneal injections. FruArg was reliably detected in the subregions of the brain tissue postinjection, indicating that it penetrates the blood–brain barrier in subnanomolar concentrations that are sufficient for its biological activity

Water-Soluble MMP‑9 Inhibitor Prodrug Generates Active Metabolites That Cross the Blood–Brain Barrier

MMP-9 plays a detrimental role in the pathology of several neurological diseases and, thus, represents an important target for intervention. The water-soluble prodrug ND-478 is hydrolyzed to the active MMP-9 inhibitor ND-322, which in turn is <i>N</i>-acetylated to the even more potent metabolite ND-364. We used a sensitive bioanalytical method based on ultraperformance liquid chromatography with multiple-reaction monitoring detection to measure levels of ND-478, ND-322, and ND-364 in plasma and brain after administration of ND-478 and the metabolites. ND-478 did not cross the blood–brain barrier, as was expected; however the active metabolites ND-322 and ND-364 distributed to the brain. The active compound after administration of either ND-478 or ND-322 is likely ND-364. ND-322 is <i>N</i>-acetylated in both brain and liver, but it is so metabolized preferentially in liver. Since <i>N</i>-acetyltransferases involved in the metabolism of ND-322 to ND-364 are polymorphic, direct administration of the <i>N</i>-acetylated ND-364 would achieve the requisite therapeutic levels in the brain

2D zymography reveals enzymatic isoforms of gelatinases of brain tissues from ZO rats.

<p>(<b>A</b>) Gelatinase activity from the brain tissues of the Zucker lean and obese rats was visualized using 1D gelatin zymography. ProMMP-9, act.MMP-9 and proMMP-2 were identified as bright bands. For the act.MMP-9, ZO vehicle-treated rats showed stronger bands than Zucker lean rats; Linagliptin ameliorates MMP-9 upregulation. (<b>B</b>) Gelatinase isoforms from ZO rats were visualized with 2D gelatin zymography. ProMMP-9 was identified as a 105-kDa single spot, act. MMP-9 as a 95-kDa single spot both with pI values between 3 and 4, as well as proMMP-2 as a 65-kDa single spot with pI value between 4 and 5. Purified gelatinases were applied on 1D zymography on the left side of the same gel for comparison. These zymograms are representative results from 4 independent experiments.</p

2D zymography reveals enzymatic isoforms of gelatinases in TBI mouse brains.

<p>Mice were sacrificed 6 h after CCI-induced TBI. In contralateral hemispheres, proMMP-9 was identified as a 105-kDa single spot with pI value between 3 and 4 and a 105-kDa streak of pI values ranging from 5.5 to 8. In lesioned hemispheres, proMMP-9 was identified as a streak with higher intensity of pI values ranging from 5.5 to 8. Traumatic brain lysate was applied on the left side of the gel for comparison. These zymograms are representative results from 4 independent experiments.</p

Comparison of manual annotation and Genie classification of cortical necrosis.

<p>After transient focal cerebral ischemia in mice, the cresyl-violet (CV)-stained brain sections were analyzed (<b>A, C</b>). For each tested region, the outlines in green, red and blue indicate manual annotations of the ischemic cortex, the non-ischemic contralateral cortex, and the cortical necrosis area, respectively. The Genie classification algorithm recognized necrotic (pink) and intact (yellow) areas within the ischemic cortex (<b>B, D</b>). When the contralateral intact cortices were analyzed, the Genie classification algorithm indicated 3.39%±0.61% (n = 18) FPR (<b>E</b>). As the manually annotated necrosis areas were analyzed by Genie algorithm, it revealed 95.99%±0.55% (n = 21) positive recognition rate (<b>F</b>). Pearson correlation coefficient between these two annotations (<b>G</b>; R = 0.957, P = 0.000, n = 32), and Bland-Altman difference plots (<b>H</b>) comparing the agreement of two measurements are shown. The red lines indicate mean and ±1.96 standard deviation. A, B, E, F: scale bar = 2 mm; C, D: scale bar = 200 μm.</p

Comparison of the manual and automated annotations of intracerebral hemorrhage.

<p>The CV-stained coronal sections after focal cerebral ischemia in mice were used to examine intracerebral hemorrhage. The outlines in red and green indicate the manual annotations of the hemorrhagic areas and the regions chose for automated algorithm analysis, respectively. When the small regions were analyzed (<b>A</b>, <b>B</b>), the two annotations showed a high degree of concordance (<b>C</b>; R = 0.943, P = 0.000, n = 30), and the Bland-Altman difference plots (<b>D</b>) indicated that the automated annotations were consistently lower than the manual annotations. The arrow (<b>A, B,</b> embedded) shows that gaps between areas of blood cells, which were not excluded by manual measurement. When the whole hemisphere of the sections with hemorrhage were analyzed (<b>E, F; G</b> and <b>H</b> show an enlarged subregion of <b>E</b> and <b>F</b>, respectively), the Pearson correlation coefficient (<b>I</b>; R = 0.335, P = 0.003, n = 75) and Bland-Altman difference plots (<b>J</b>) showed low concordance and large difference between the manual and automated annotations. The red lines (in <b>D</b> and <b>J</b>) indicate mean and ±1.96 standard deviation. A, B, G, H: scale bar = 100 μm; E, F: scale bar = 2 mm.</p