Animals ; Antioxidants ; pharmacology ; Fatigue ; prevention & control ; Male ; Mice ; Physical Exertion ; physiology ; Random Allocation ; Recombinant Proteins ; genetics ; metabolism ; pharmacology ; Superoxide Dismutase ; genetics ; metabolism ; pharmacology ; Swimming ; physiology

Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage. However, the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells, thus protecting tumor cells and consequently reducing the efficacy of radiotherapy. There are active matrix metalloproteinase (MMP)-2 or MMP-9 in most tumor cellular microenvironments. Therefore, a fusion protein containing an MMP-2/9 cleavable substrate peptide X, a cell-penetrating peptide R9, a glutathione S-transferase (GST), and a human Cu, Zn superoxide dismutase (SOD1), was designed and named GST-SOD1-X-R9. In the tumor microenvironment, GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9, thereby achieving selected entering normal cells. The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1. The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained, and soluble expression of the fusion protein was achieved. GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography. The molecular weight of the fusion protein was approximately 47 kDa, consistent with the theoretical value. The SOD and GST activities were 2 954 U/mg and 328 U/mg, respectively. Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions. The fusion protein could be partially digested by collagenase Ⅳ in solution. Subsequently, the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells. Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition. While under the 3D culture model, the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily. GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells, but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably. This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells.
Ammonium Sulfate ; Antioxidants ; Cell-Penetrating Peptides/pharmacology* ; Endopeptidases ; Glutathione Transferase/metabolism* ; Humans ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Recombinant Fusion Proteins ; Recombinant Proteins ; Superoxide Dismutase/metabolism* ; Superoxide Dismutase-1

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons in the brain and spinal cord. One important aspect of ALS pathogenesis is superoxide dismutase 1 (SOD1) mutant-mediated mitochondrial toxicity, leading to apoptosis in neurons. This study aimed to evaluate the neural protective synergistic effects of ginsenosides Rg1 (G-Rg1) and conditioned medium (CM) on a mutational SOD1 cell model, and to explore the underlying mechanisms. We found that the contents of nerve growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor significantly increased in CM after human umbilical cord mesenchymal stem cells (hUCMSCs) were exposed to neuron differentiation reagents for seven days. CM or G-Rg1 decreased the apoptotic rate of SOD1^G93A-NSC34 cells to a certain extent, but their combination brought about the least apoptosis, compared with CM or G-Rg1 alone. Further research showed that the anti-apoptotic protein Bcl-2 was upregulated in all the treatment groups. Proteins associated with mitochondrial apoptotic pathways, such as Bax, caspase 9 (Cas-9), and cytochrome c (Cyt c), were downregulated. Furthermore, CM or G-Rg1 also inhibited the activation of the nuclear factor-kappa B (NF-κB) signaling pathway by reducing the phosphorylation of p65 and IκBα. CM/G-Rg1 or their combination also reduced the apoptotic rate induced by betulinic acid (BetA), an agonist of the NF-κB signaling pathway. In summary, the combination of CM and G-Rg1 effectively reduced the apoptosis of SOD1^G93A-NSC34 cells through suppressing the NF-κB/Bcl-2 signaling pathway (Fig. 1 is a graphical representation of the abstract).
Humans ; NF-kappa B/metabolism* ; Ginsenosides/pharmacology* ; Amyotrophic Lateral Sclerosis/genetics* ; Culture Media, Conditioned/pharmacology* ; Superoxide Dismutase-1 ; Neurodegenerative Diseases ; Neurons/metabolism* ; Apoptosis

AIMTo investigate the antioxidant mechanisms of propylene glycol mannate sulfate (PGMS) in hyperlipidemic rats.

METHODSMale Wistar rats were given high lipid emulsion diet to establish hyperlipidemic model. PGMS was given every day at different doses (37.8 and 75.6 mg.kg-1, ig) to hyperlipidemic rats for three weeks. In addition, diethyldithiocarbamate (DDC) was given 200 mg.kg-1.3 d-1 (i.p.) to inhibit SOD activity. Then, the MDA content was examined using TBA method to show the oxidation level, and the activities of SOD, GSH-Px and CAT were examined following the kit protocols to indicate the capability of eliminating OFR. RT-PCR was applied to study the expression of Cu, Zn-SOD mRNA in rat liver.

RESULTSThe MDA content of PGMS treatment groups decreased markedly compared with hyperlipidemic group, and the activities of SOD, GSH-Px and CAT increased distinctly. Cu, Zn-SOD mRNA expression was significantly increased by PGMS treatment. Furthermore, the application of DDC(the SOD inhibitor) reduced total SOD activity and Cu, Zn-SOD mRNA expression induced by PGMS, and the content of MDA increased correspondingly.

CONCLUSIONPGMS can induce the activities of antioxidant enzymes and the mRNA expression of Cu, Zn-SOD, which contribute to the elimination of oxygen free radical. This may explain the molecular mechanism of antioxidant effects of PGMS.

Animals ; Antioxidants ; pharmacology ; Catalase ; metabolism ; Free Radical Scavengers ; pharmacology ; Hyperlipidemias ; enzymology ; metabolism ; Liver ; enzymology ; metabolism ; Male ; Malondialdehyde ; metabolism ; Propylene Glycols ; pharmacology ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Superoxide Dismutase ; biosynthesis ; genetics ; metabolism

OBJECTIVETo investigate the antagonistic effect and mechanism of the effect of cyproheptadine (Cyp) on endotoxic shock in rats.

METHODSEndotoxic shock was produced in rats by i.v. injection of lipopolysaccharides (LPS) (5 mg/kg). Tumor necrosis factor (TNF(alpha)) mRNA expression was assessed by Northern blot. Plasma TNF(alpha) content was measured by radioimmunoassay. Plasma superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured. The intracellular free calcium concentration ([Ca(2+)](i)) in single endothelial cells was determined by laser scanning confocal microscopy (LSCM).

RESULTSCyp 5 mg/kg injected immediately after i.v. LPS raised the mean arterial blood pressure (MABP) of shocked rats and improved their 24 h survival rate. Meanwhile, Cyp markedly decreased TNF(alpha) mRNA levels in rat liver (18 +/- 10 vs. LPS + saline 38 +/- 10, P < 0.01) as well as plasma TNF(alpha) content [(7.8 +/- 2.4) microg/L vs. LPS + saline (21.5 +/- 3.2) microg/L, P < 0.01)]. It enhanced plasma SOD activity [(1037.2 +/- 112.8) NU/L vs LPS + saline (615.4 +/- 92.6) NU/L, P < 0.01], reduced the MDA content [(5.2 +/- 1.1) micromol/L vs. LPS + saline (9.8 +/- 1.5) micromol/L, P < 0.01], and inhibited TNF(alpha)-induced [Ca(2+)](i) elevation.

CONCLUSIONCyp exerts an anti-endotoxic shock effect by inhibiting TNF(alpha) gene expression, enhancing SOD activity, reducing lipid peroxidation, and preventing [Ca(2+)](i) overload.

Animals ; Cyproheptadine ; pharmacology ; Histamine H1 Antagonists ; pharmacology ; Malondialdehyde ; blood ; Rats ; Rats, Wistar ; Shock, Septic ; drug therapy ; metabolism ; Superoxide Dismutase ; blood ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of losartan (an angiotensin II type I receptor antagonist) on endothelial cells exposed to high glucose in vitro and related mechanism.

METHODSVascular endothelial cells of human umbilical vein were cultured in media with high glucose levels. The activities of SOD and CAT, the level of MDA were measured by spectrophotometry in the conditioned media of endothelial cells, the VEGF mRNA expression was performed using semi-quantitative reverse transcription PCR (RT-PCR) in the cell lysates, and the protein expression of VEGF was examined by enzyme-linked immunosorbent assay (ELISA) in the supernatants of cultured cells.

RESULTWhen endothelial cells were cultured in high glucose, the activities of SOD and CAT were significantly decreased, but the level of MDA was markedly increased. However, the high glucose-induced effects were inhibited by losartan. The application of high glucose upregulated the mRNA and protein expression of VEGF in endothelial cells, which was also attenuated by losartan.

CONCLUSIONHigh glucose disrupts the oxidative equilibrium and increases the expression of VEGF in endothelial cells, which can be inhibited by losartan.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Glucose ; pharmacology ; Humans ; Losartan ; pharmacology ; Peroxidase ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Superoxide Dismutase ; metabolism ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factors ; biosynthesis ; genetics

AIMTo investigate the protective effect of melatonin (MT) on lung tissues during acute lung injury (ALI) in rats and its possible mechanism.

METHODSAll rats were randomly divided into four groups: control group, lipopolysaccharide (LPS) group, dexamethasone (DEX) and MT treatment group. Myeloperoxidase (MPO) activity, superoxide dismutase (SOD) activity and malonaldehyde (MDA) content of lung tissues were detected at 3, 6 and 12 h after intratracheal instillation in each group. In addition, the expression of intercellular adhesion molecule-1 (ICAM-1) were observed through immunohistochemistry staining in lung tissues.

RESULTSCompared with control group, SOD activity decreased significantly in LPS group (P < 0.01), but MPO activity,MDA content and the expression of ICAM-1 increased obviously (P < 0.01). The administration of MT and DEX mitigated above changes significantly (P < 0.05 or P < 0.01).

CONCLUSIONMT possessed protective effect on lung tissues during ALI through scavenging free radicals and inhibiting the expression of ICAM-1 probably.

Acute Lung Injury ; chemically induced ; metabolism ; physiopathology ; Animals ; Antioxidants ; pharmacology ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Lipopolysaccharides ; Male ; Melatonin ; pharmacology ; Peroxidase ; metabolism ; Protective Agents ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

This study aims to investigate the effect and mechanism of total triterpenes of Euphorbium in treating rheumatoid arthritis(RA). The rat model of RA was established with Freund's complete adjuvant(FCA). Male rats were randomly assigned into control, model, Tripterygium glycosides(7.5 mg·kg~(-1)), and low-, medium-, and high-dose total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1), respectively) groups, with 10 rats in each group. In other groups except the control group, 0.2 mL FCA was injected into the right hind toe. Rats in the intervention groups were administrated with corresponding drugs by gavage, and the control group and the model group with the same volume of 0.5% CMC-Na solution once a day. During the treatment period, the swelling degree of the hind paw was measured and the arthritis was scored until day 30. At the end of drug administration, the pathological changes of the joint tissue were observed by hematoxylin-eosin staining. The content of malondialdehyde(MDA), glutathione(GSH), and Fe~(2+) and the activity of superoxide dismutase(SOD) in the joint tissue were measured by biochemical colorimetry. RT-PCR was performed to determine the mRNA levels of nuclear factor erythroid 2-related factor 2(Nrf2), glutathione peroxidase 4(GPX4), and acyl-CoA synthetase long chain family member 4(ACSL4) in the joint tissue. Western blot was employed to determine the protein levels of Nrf2, Kelch-like ECH-associated protein 1(Keap1), heme oxygenase-1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), SOD2, GPX4, and ACSL4 in the joint tissue. The results showed that the treatment with Tripterygium glycosides(7.5 mg·kg~(-1)) and total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1)) alleviated the swelling degree of bilateral hind limbs, decreased the arthritis score, reduced joint tissue lesions and the content of MDA and Fe~(2+) in the joint tissue, and increased GSH content and SOD activity. Furthermore, the interventions up-regulated the protein and mRNA levels of Nrf2 and GPX4, down-regulated the protein and mRNA levels of ACSL4, and up-regulated the protein levels of Keap1, NQO1, HO-1, and SOD2 in the Nrf2/HO-1/GPX4. In summary, the total triterpenes of Euphorbium can treat RA by inhibiting lipid peroxidation and abnormal ferroptosis, which may involve the Nrf2/HO-1/GPX4 signaling pathway.
Rats ; Male ; Animals ; Rats, Sprague-Dawley ; NF-E2-Related Factor 2/metabolism* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Triterpenes/pharmacology* ; Oxidative Stress ; Arthritis, Rheumatoid/genetics* ; Glutathione ; Superoxide Dismutase/metabolism* ; Glycosides/pharmacology* ; RNA, Messenger/metabolism*

This study aimed to investigate the biological effects and underlying mechanisms of the total ginsenosides from Panax ginseng stems and leaves on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in mice. Sixty male C57BL/6J mice were randomly divided into a control group, a model group, the total ginsenosides from P. ginseng stems and leaves normal administration group(61.65 mg·kg~(-1)), and low-, medium-, and high-dose total ginsenosides from P. ginseng stems and leaves groups(15.412 5, 30.825, and 61.65 mg·kg~(-1)). Mice were administered for seven continuous days before modeling. Twenty-four hours after modeling, mice were sacrificed to obtain lung tissues and calculate lung wet/dry ratio. The number of inflammatory cells in bronchoalveolar lavage fluid(BALF) was detected. The levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in BALF were detected. The mRNA expression levels of IL-1β, IL-6, and TNF-α, and the levels of myeloperoxidase(MPO), glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), and malondialdehyde(MDA) in lung tissues were determined. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in lung tissues. The gut microbiota was detected by 16S rRNA sequencing, and gas chromatography-mass spectrometry(GC-MS) was applied to detect the content of short-chain fatty acids(SCFAs) in se-rum. The results showed that the total ginsenosides from P. ginseng stems and leaves could reduce lung index, lung wet/dry ratio, and lung damage in LPS-induced ALI mice, decrease the number of inflammatory cells and levels of inflammatory factors in BALF, inhibit the mRNA expression levels of inflammatory factors and levels of MPO and MDA in lung tissues, and potentiate the activity of GSH-Px and SOD in lung tissues. Furthermore, they could also reverse the gut microbiota disorder, restore the diversity of gut microbiota, increase the relative abundance of Lachnospiraceae and Muribaculaceae, decrease the relative abundance of Prevotellaceae, and enhance the content of SCFAs(acetic acid, propionic acid, and butyric acid) in serum. This study suggested that the total ginsenosides from P. ginseng stems and leaves could improve lung edema, inflammatory response, and oxidative stress in ALI mice by regulating gut microbiota and SCFAs metabolism.
Mice ; Male ; Animals ; Ginsenosides/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6 ; Panax/genetics* ; Lipopolysaccharides/adverse effects* ; Gastrointestinal Microbiome ; RNA, Ribosomal, 16S ; Mice, Inbred C57BL ; Acute Lung Injury/genetics* ; Lung/metabolism* ; Superoxide Dismutase/metabolism* ; Plant Leaves/metabolism* ; RNA, Messenger

AIMTo study the effect of propylene glycol mannate sulfate (PGMS) on induction of CuZn-SOD.

METHODSWistar rats were given PGMS p.o. at different doses (0, 18.9, 37.8 and 75.6 mg.kg-1.d) for ten days. Then the rats were sacrificed and the total RNA was extracted from the livers. The total RNA samples were loaded on a 1% agarose gel to detect the quality of total RNA. RT-PCR was applied to study the expression of CuZn-SOD mRNA in rat livers. The amplified products were detected by the 1.5% agarose gel electrophoresis. Simultaneously, the CuZn-SOD activities in rat liver were determined by nitrite method.

RESULTSThe total RNA extracted from rat livers was integrated without being decomposed by RNase. The level of CuZn-SOD mRNA of the high-dosage group (75.6 mg.kg-1.d) was higher than that of the control group (0 mg.kg-1.d) (P < 0.01); the CuZn-SOD activities of the high-dosage group were significantly higher than those of the control group (P < 0.001) and the CuZn-SOD activities of the middle- (37.8 mg.kg-1.d) and low-dosage groups (18.9 mg.kg-1.d) were higher than those of the control group (P < 0.01).

CONCLUSIONPGMS can increase the CuZn-SOD activities as well as CuZn-SOD on mRNA level. Therefore, it is possible for PGMS to counteract Atherosclerosis (AS) by inducing the expression of CuZn-SOD.

Animals ; Dose-Response Relationship, Drug ; Free Radical Scavengers ; pharmacology ; In Vitro Techniques ; Liver ; drug effects ; metabolism ; Male ; Propylene Glycols ; pharmacology ; RNA, Messenger ; biosynthesis ; drug effects ; genetics ; Rats ; Rats, Wistar ; Superoxide Dismutase ; biosynthesis ; genetics ; metabolism