OBJECTIVE

The aim of this study was to investigate the protective effects of Lactobacillus brevis BIOTECH 1766 against oxidative damage in the brain, liver, and kidneys induced by aluminum (Al) poisoning in ICR mice.

Twenty mice were divided into four groups (n = 5): (I) control, (II) Al, (III) citric acid (CA), and (IV) L. brevis BIOTECH 1766 group. A 14-day treatment period was implemented, wherein groups I and II received sterile water, while groups III and IV received 10 mg/kg bw of CA and 1 x 109 cfu/kg bw of L. brevis BIOTECH 1766, respectively. On day 15, all except the control group received a single oral dose of 1438 mg/kg bw of AlCl3. 6H2O. After 24 h, mice were euthanized to collect the brain, liver, and kidneys for the oxidative stress marker analyses and histopathological examination.

Acute intoxication of Al led to a significant increase in tissue malondialdehyde (MDA) and a significant decrease in the tissue's reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD). Mice pretreated with CA or L. brevis BIOTECH 1766 have markedly reduced CAT activity in the liver, and SOD in all three organs. Extensive organ injuries were also prevented by CA and L. brevis BIOTECH 1766 pretreatment, with the latter providing better protection against liver damage.

The findings showed that L. brevis BIOTECH 1766 provides a protective effect against acute Al poisoning in mice by ameliorating oxidative damage in the brain, liver, and kidneys.

The effect and mechanism of Heixiaoyao Powder on the polarization of microglia(MG) in APP/PS1 double transgenic mice were explored based on NADPH oxidase 2(NOX2)/reactive oxygen species(ROS)/nuclear factor kappaB(NF-κB) signaling pathway. Fifty 4-month-old male APP/PS1 mice were randomly divided into a model group, an MCC950 group(10 mg·kg~(-1)), and low-, medium-, and high-dose Heixiaoyao Powder groups(6.45, 12.89, and 25.78 g·kg~(-1)). Thirty male C57BL/6J mice of the same age and strain were randomly divided into a blank group, a blank + intragastric intervention group, and a blank + intraperitoneal injection group. Drug intervention lasted 90 days. Morris water maze test was used to detect learning and cognitive ability. Nissl staining and transmission electron microscopy were used to observe the pathological morphology and ultrastructure of hippocampal neurons. Immunofluorescence was used to detect the positive expression of M1-type marker CD16/32~+/Iba-1~+, M2-type marker CD206~+/Iba-1~+ of MG and the expression of hippocampal ROS. The colorimetric method was used to detect the content of malondialdehyde(MDA) and superoxide dismutase(SOD) in the hippocampus. Enzyme linked immunosorbent assay(ELISA) was used to detect the levels of inflammatory factors, including interleukin-6(IL-6), interleukin-8(IL-8), and tumor necrosis factor-α(TNF-α), in the hippocampus. Western blot was used to detect the protein expression of β-amyloid protein(Aβ), Iba-1, CD16/32, CD206, NOX2, NF-κB, p-NF-κB, NF-κB inhibitor alpha(IκBα), and p-IKBα in the hippocampus. The results showed that as compared with the blank group, the model group showed prolonged target quadrant movement distance and escape latency(P<0.01), shortened target quadrant retention time and percentage(P<0.01), disorganized neuronal cells with swelling, nuclear disappearance or bias, reduced number of cells, dissolved or absent Nissl bodies, and a clear area in the cytoplasm, damaged and shrunk cell membrane with abnormal cell morphology, few organelles in the cytoplasm, reduced and swollen mitochondria, increased MG M1-type marker CD16/32~+/Iba-1~+(P<0.01), decreased M2-type marker CD206~+/Iba-1~+(P<0.01), increased ROS activity and MDA content(P<0.01), decreased SOD level(P<0.01), elevated inflammatory factors IL-6, IL-8, and TNF-α(P<0.01), up-regulated protein expression and phosphorylation of Aβ, CD16/32, Iba-1, NOX2, NF-κB, and IKBα(P<0.01), and down-regulated CD206(P<0.01). There was no statistically significant difference between the blank group, the blank + intragastric intervention group, and the blank + intraperitoneal injection group. After the intervention of Heixiaoyao Powder, the Heixiaoyao Powder groups showed shortened target quadrant movement distance and escape latency(P<0.01), prolonged target quadrant retention time and percentage(P<0.01), increased and neatly arranged cells with relieved swelling, increased Nissl bodies, regular cell morphology, and intact cell membrane, relieved swelling of mitochondria, slightly expanded endoplasmic reticulum, decreased CD16/32~+/Iba-1~+(P<0.05 or P<0.01), increased CD206~+/Iba-1~+(P<0.01), decreased ROS activity and MDA content(P<0.01), increased SOD level(P<0.01), decreased content of inflammatory factors IL-6, IL-8, and TNF-α(P<0.01), down-regulated protein expression and phosphorylation of Aβ, CD16/32, Iba-1, NOX2, NF-κB, and IKBα(P<0.01), and up-regulated CD206(P<0.01). In conclusion, Heixiaoyao Powder can alleviate neuronal damage and improve the learning and memory abilities of APP/PS1 mice. The mechanism of action may be related to the inhibition of NOX2/ROS/NF-κB signaling pathway, regulating the polarization of MG, increasing the expression of M2 type, inhibiting the expression of M1 type, and reducing the release of inflammatory factor.
Mice ; Male ; Animals ; NF-kappa B/genetics* ; Microglia ; Reactive Oxygen Species ; Interleukin-8 ; Powders ; Tumor Necrosis Factor-alpha ; Interleukin-6 ; Mice, Inbred C57BL ; Signal Transduction ; Mice, Transgenic ; Superoxide Dismutase

This study aimed to investigate the protective effect and underlying mechanism of Mailuo Shutong Pills(MLST) on posterior limb swelling caused by femur fracture in rats. The rats were randomly divided into a sham operation group, a model group, a low-dose MLST group(1.8 g·kg~(-1)·d~(-1)), a high-dose MLST group(3.6 g·kg~(-1)·d~(-1)), and a positive drug group(60 mg·kg~(-1)·d~(-1) Maizhiling Tablets). The femur in the sham operation group was exposed and the wound was sutured, while the other four groups underwent mechanical damage to cause femur fracture. The rats were treated with corresponding drugs by gavage 7 days before modeling and 5 days after modeling, while those in the sham operation group and the model group were given an equivalent dose of distilled water by gavage. Hematoxylin-eosin(HE) staining was used to detect the pathological injury of the posterior limb muscle tissues in rats, and the degree of hind limb swelling was measured. The enzyme-linked immunosorbent assay(ELISA) kit was used to detect the expression levels of interleukin-6(IL-6), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) in the serum of rats in each group. The activity of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and glutathione peroxidase(GSH-Px) in rat serum was also measured. Western blot was used to detect the protein expression levels of heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), and nuclear transcription factor E2-related factor 2(Nrf2) in rat posterior limb muscle tissues. The changes in the intestinal flora and intestinal metabolites in rats were detected by 16S rDNA sequencing and ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), respectively, to explore the underlying mechanism of MLST in treating posterior limb swelling caused by femur fracture in rats. Compared with the model group, MLST significantly improved the degree of posterior limb swelling in rats, reduced the levels of serum inflammatory factors, and alleviated oxidative stress injury. The HE staining results showed that the inflammatory infiltration in the posterior limb muscle tissues of rats in the MLST groups was significantly improved. Western blot results showed that MLST significantly increased the protein expression of HO-1, NQO1, and Nrf2 in rat posterior limb muscle tissues compared with the model group. The 16S rDNA sequencing results showed that MLST improved the disorder of intestinal flora in rats after femur fracture. The UPLC-MS/MS results showed that MLST significantly affected the bile acid biosynthesis and metabolism pathway in the intestine after femur fracture, and the Spearman analysis confirmed that the metabolite deoxycholic acid involved in bile acid biosynthesis was positively correlated with the abundance of Turicibacter. The metabolite cholic acid was positively correlated with the abundance of Papilibacter, Staphylococcus, and Intestinimonas. The metabolite lithocholic acid was positively correlated with Papilibacter and Intestinimonas. The above results indicated that MLST could protect against the posterior limb swelling caused by femur fracture in rats. This protective effect may be achieved by improving the pathological injury of the posterior limb muscle, reducing the expression levels of inflammatory and oxidative stress-related factors in serum, reducing the oxidative injury of the posterior limb muscle, improving intestinal flora, and balancing the biosynthesis of bile acids in the intestine.
Rats ; Animals ; NF-E2-Related Factor 2/metabolism* ; Gastrointestinal Microbiome ; Chromatography, Liquid ; Multilocus Sequence Typing ; Tandem Mass Spectrometry ; Oxidative Stress ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Femur ; Bile Acids and Salts ; DNA, Ribosomal ; 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 aims to investigate the effects of baicalein(BAI) on lipopolysaccharide(LPS)-induced human microglial clone 3(HMC3) cells, with a focus on suppressing inflammatory responses and elucidating the potential mechanism underlying the therapeutic effects of BAI on ischemic stroke via modulating the cAMP-PKA-NF-κB/CREB pathway. The findings have significant implications for the application of traditional Chinese medicine in treating cerebral ischemic diseases. First, the safe dosage of BAI was screened, and then an inflammation model was established with HMC3 cells by induction with LPS for 24 h. The cells were assigned into a control group, a model group, and high-, medium-, and low-dose(5, 2.5, and 1.25 μmol·L~(-1), respectively) BAI groups. The levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in cell extracts, as well as the levels of interleukin-1β(IL-1β), IL-6, tumor necrosis factor-α(TNF-α), and cyclic adenosine monophosphate(cAMP) in the cell supernatant, were measured. Western blot was performed to determine the expression of protein kinase A(PKA), phosphorylated cAMP-response element binding protein(p-CREB), and nuclear factor-kappa B p65(NF-κB p65). Hoechst 33342/PI staining was employed to assess cell apoptosis. High and low doses of BAI were used for treatment in the research on the mechanism. The results revealed that BAI at the concentrations of 10 μmol·L~(-1) and below had no impact on normally cultured HMC3 cells. LPS induction at 200 ng·mL~(-1) for 24 h reduced the SOD activity and increased the MDA content in HMC3 cells. However, 5, 2.5, and 1.25 μmol·L~(-1) BAI significantly increased the SOD activity and 5 μmol·L~(-1) BAI significantly decreased the MDA content. In addition, BAI ameliorated the M1 polarization of HMC3 cells induced by LPS, as indicated by cellular morphology. The results of ELISA demonstrated that BAI significantly lowered the levels of TNF-α, IL-1β, IL-6, and cAMP in the cell supernatant. Western blot revealed that BAI up-regulated the protein levels of PKA and p-CREB while down-regulating the expression of NF-κB p65. Hoechst 33342/PI staining results indicated that BAI mitigated the apoptosis of HMC3 cells. Overall, the results indicated that BAI had protective effects on the HMC3 cells induced by LPS, and could inhi-bit inflammatory response and improve cell apoptosis, which might be related to the regulation of the cAMP-PKA-NF-κB/CREB pathway.
Humans ; NF-kappa B/metabolism* ; Microglia ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Lipopolysaccharides/pharmacology* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Superoxide Dismutase/metabolism*

This study aims to investigate the mechanism of acacetin in protecting rats from cerebral ischemia-reperfusion injury via the Toll-like receptor 4(TLR4)/NOD-like receptor protein 3(NLRP3) signaling pathway. Wistar rats were randomized into sham, model, low-and high-dose acacetin, and nimodipine groups, with 10 rats in each group. The rat model of middle cerebral artery occlusion(MCAO) was established with the improved suture method in other groups except the sham group. The neurological deficit score and cerebral infarction volume of each group were evaluated 24 h after modeling. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1β(IL-1β), IL-6, tumor necrosis factor-α(TNF-α), malondialdehyde(MDA), supe-roxide dismutase(SOD), and glutathione(GSH). Western blot was employed to determine the expression levels of B-cell lymphonoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and TLR4/NLRP3 signaling pathway-related proteins(TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1β, and cleaved IL-1β) in the rat brain tissue. Hematoxylin-eosin(HE) staining was employed to reveal the histopathological changes in the ischemic area. Compared with the sham group, the modeling of MCAO increased the neurological deficit score and cerebral infarction volume, elevated the IL-1β, IL-6, TNF-α, and MDA levels and lowered the SOD and GSH levels in the brain tissue(P<0.05). Compared with the MCAO model group, low-and high-dose acacetin and nimodipine decreased the neurological deficit score and cerebral infarction volume, lowered the IL-1β, IL-6, TNF-α, and MDA levels and elevated the SOD and GSH levels in the brain tissue(P<0.05). Compared with the sham group, the model group showed up-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1β, and cleaved IL-1β and down-regulated protein level of Bcl-2 in the brain tissue(P<0.05). Compared with the MCAO model group, the acacetin and nimodipine groups showed down-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1β, and cleaved IL-1β and up-regulated protein level of Bcl-2 in the brain tissue(P<0.05). In conclusion, acacetin regulates the TLR4/NLRP3 signaling pathway to inhibit neuroinflammatory response and oxidative stress, thus exerting the protective effect on cerebral ischemia-reperfusion injury in rats.
Rats ; Animals ; NF-kappa B/metabolism* ; bcl-2-Associated X Protein ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Rats, Sprague-Dawley ; Caspase 1/metabolism* ; Toll-Like Receptor 4/metabolism* ; Nimodipine/pharmacology* ; Interleukin-6 ; Rats, Wistar ; Signal Transduction ; Infarction, Middle Cerebral Artery ; Reperfusion Injury/prevention & control* ; Superoxide Dismutase/metabolism*

This paper aims to investigate the protective effect and mechanism of Astragalus membranaceus and Angelica sinensis before and after compatibility against triptolide(TP)-induced hepatotoxicity. The experiment was divided into a blank group, model group, Astragalus membranaceus group, Angelica sinensis group, and compatibility groups with Astragalus membranaceus/Angelica sinensis ratio of 1∶1, 2∶1, and 5∶1. TP-induced hepatotoxicity model was established, and corresponding drug intervention was carried out. The levels of alanine transaminase(ALT), aspartate transaminase(AST), and alkaline phosphatase(ALP) in serum were detected. Pathological injuries of livers were detected by hematoxylin-eosin(HE) staining. The levels of malondialdehyde(MDA), superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), and reduced glutathione(GSH) in the liver were measured. Wes-tern blot method was used to detect the expression of nuclear factor erythroid 2-related factor 2(Nrf2), Kelch-like ECH-associated protein 1(Keap1), peroxisome proliferator-activated receptor gamma, coactivator-1 alpha(PGC-1α), heme oxygenase-1(HO-1), and NAD(P)H quinone dehydrogenase 1(NQO1) in livers. Immunofluorescence was used to detect the expression of Nrf2 and PGC-1α in livers. The results indicated that Astragalus membranaceus/Angelica sinensis ratio of 2∶1 and 5∶1 could significantly reduce the levels of serum AST, ALT, and ALP, improve the pathological damage of liver tissue, increase the levels of GSH and GSH-Px, and reduce the content of MDA in liver tissue. Astragalus membranaceus/Angelica sinensis ratio of 1∶1 and 2∶1 could significantly improve the level of SOD. Astragalus membranaceus and Angelica sinensis before and after compatibility significantly increased the protein expression of HO-1 and NQO1, improved the protein expression of Nrf2 and PGC-1α, and decreased the protein expression of Keap1 in liver tissue. The above results confirmed that the compatibility of Astragalus membranaceus and Angelica sinensis had antioxidant effects by re-gulating Keap1/Nrf2/PGC-1α, and the Astragalus membranaceus/Angelica sinensis ratio of 2∶1 and 5∶1 had stronger antioxidant effect and significantly reduced TP-induced hepatoto-xicity.
Humans ; Astragalus propinquus ; Angelica sinensis ; NF-E2-Related Factor 2/metabolism* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Antioxidants/pharmacology* ; Chemical and Drug Induced Liver Injury/prevention & control* ; Superoxide Dismutase/metabolism* ; Oxidative Stress ; Diterpenes ; Epoxy Compounds ; Phenanthrenes

This study aimed to investigate the effect and underlying mechanism of Poria cocos polysaccharides(PCP) on myocardial cell apoptosis in the rat model of myocardial ischemia-reperfusion injury(MI/RI). Male SPF-grade SD rats were randomly divided into a sham group(saline), a model group(saline), low-and high-dose PCP groups(100 and 200 mg·kg~(-1)), and a fasudil group(10 mg·kg~(-1)), with 16 rats in each group. Except for the sham group, the other four groups underwent left anterior descending coronary artery ligation for 30 min followed by reperfusion for 2 h to establish the MI/RI model. The myocardial infarct area was assessed by TTC staining. Histological changes were observed through HE staining. Myocardial cell apoptosis was evaluated using TUNEL staining. Serum lactate dehydrogenase(LDH), creatine kinase MB(CK-MB), interleukin-1β(IL-1β) and IL-18 levels, myocardial superoxide dismutase(SOD) activity and malondialdehyde(MDA) levels were detected by ELISA. Protein expression of B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), cleaved caspase-3, Ras homolog gene A(RhoA), myosin phosphatase target subunit 1(MYPT-1), phosphorylated MYPT-1(p-MYPT-1), and Rho-associated coiled-coil forming kinase 1(ROCK 1) were measured by Western blot. Pathological staining of myocardial tissue revealed that in the model group, there was focal necrosis of myocardial tissue, myocardial cell swelling, unclear boundaries, and neutrophil infiltration. These pathological changes were alleviated in the low-and high-dose PCP groups and the fasudil group. Compared with the model group, the low-and high-dose PCP groups and the fasudil group showed significantly reduced myocardial infarct area and myocardial cell apoptosis rate. Compared with the sham group, the model group exhibited elevated serum LDH, CK-MB, IL-1β and IL-18 levels, increased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and decreased myocardial SOD levels and Bcl-2 protein expression. Compared with the model group, the PCP groups and the fasudil group showed lowered serum LDH, CK-MB, IL-1β and IL-18 levels, decreased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and increased myocardial SOD levels and Bcl-2 protein expression. PCP exhibited a certain preventive effect on myocardial tissue pathological damage and myocardial cell apoptosis in MI/RI rats, possibly related to the inhibition of the Rho-ROCK signaling pathway activation, thereby reducing oxidative stress and inflammatory responses.
Rats ; Male ; Animals ; Myocardial Reperfusion Injury/drug therapy* ; bcl-2-Associated X Protein/metabolism* ; Rats, Sprague-Dawley ; Caspase 3/metabolism* ; Interleukin-18 ; Wolfiporia ; Signal Transduction ; Myocardial Infarction/drug therapy* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Creatine Kinase, MB Form ; Apoptosis ; Polysaccharides/pharmacology* ; Superoxide Dismutase/metabolism* ; 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives*

This study explored the effect and mechanism of Maiwei Yangfei Decoction(MWYF) on pulmonary fibrosis(PF) mice. MWYF was prepared, and its main components were detected by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-MS/MS). Male C57BL/6J mice were randomly divided into a control group, a model group, a pirfenidone(PFD) group, and low-, medium-, and high-dose MWYF groups, with 10 mice in each group. The PF model was induced in mice except for those in the control group by intratracheal instillation of bleomycin(BLM), and model mice were treated with saline or MWYF or PFD by gavage the next day. The water consumption, food intake, hair, and activity of mice were observed daily. The pathological changes in lung tissues were observed by hematoxylin-eosin(HE) staining, Masson staining, and CT scanning. The level of hydroxyproline(HYP) in lung tissues was detected by alkaline hydrolysis. Immunohistochemistry was used to observe the expression of collagen type Ⅲ(COL3) and fibronectin. The mRNA expression levels of α-smooth muscle actin(α-SMA), type Ⅰ collagen α1(COL1α1), COL3, and vimentin were detected by reverse transcription real-time fluorescence quantitative polymerase chain reaction(RT-qPCR). Superoxide dismutase(SOD) and malondialdehyde(MDA) kits were used to detect oxidative stress indicators in lung tissues and serum. The nuclear translocation of nuclear factor E2-related factor 2(Nrf2) protein was detected by immunofluorescence. The protein and mRNA expression levels of Nrf2, catalase(CAT), and heme oxygenase 1(HO-1) in lung tissues were detected by Western blot and RT-qPCR. Twelve chemical components were detected by UPLC-MS/MS. Animal experiments showed that MWYF could improve alveolar inflammation, collagen deposition, and fibrosis in PF mice, increase body weight of mice, and down-regulate the expression of fibrosis indexes such as HYP, α-SMA, COL1α1, COL3, fibronectin, and vimentin in lung tissues. In addition, MWYF could potentiate the activity of SOD in lung tissues and serum of PF mice, up-regulate the expression level of Nrf2, and promote its transfer to the nucleus, up-regulate the levels of downstream antioxidant target genes CAT and HO-1, and then reduce the accumulation of lipid metabolite MDA. In summary, MWYF can significantly improve the pathological damage and fibrosis of lung tissues in PF mice, and its mechanism may be related to the activation of the Nrf2 pathway to regulate oxidative stress.
Mice ; Male ; Animals ; Pulmonary Fibrosis/chemically induced* ; NF-E2-Related Factor 2/metabolism* ; Fibronectins/metabolism* ; Vimentin/metabolism* ; Chromatography, Liquid ; Mice, Inbred C57BL ; Tandem Mass Spectrometry ; Oxidative Stress ; Superoxide Dismutase/metabolism* ; RNA, Messenger/metabolism*

This study observed the effects of Guiqi Yiyuan Ointment(GQYY) on the left lung subjecting to bystander effect of right lung injury induced by ~(12)C~(6+) beam in rats and decipher the underlying mechanism from NOD-like receptor protein 3(NLRP3)/apoptosis-associated speck-like protein containing a CARD(ASC)/cysteinyl aspartate specific proteinase-1(caspase-1) pathway. Wistar rats were randomized into 7 groups: blank, model, inhibitor [200 mg·kg~(-1), N-acetylcysteine(NAC)], western drug [140 mg·kg~(-1) amifostine(AMI)], and high-, medium-, and low-dose(4.8, 2.4, and 1.2 g·kg~(-1), respectively) GQYY groups. The model of bystander effect damage was established by 4 Gy ~(12)C~(6+) beam irradiation of the right lung(with the other part shielded by a lead plate). The pathological changes in the lung tissue, the level of reactive oxygen species(ROS) in the lung tissue, and the levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in the serum were observed and measured in each group. Furthermore, the mRNA and protein levels of NLRP3, ASC, caspase-1, and phosphorylated nuclear factor-κB p65(p-NF-κB p65)/nuclear factor-κB p65(NF-κB p65) were determined. Compared with the blank group, the model group showed thickened alveolar wall, narrowed alveolar cavity, and presence of massive red blood cells and inflammatory infiltration in the alveolar wall and alveolar cavity. In addition, the model group showed elevated ROS levels in both left and right lungs, elevated MDA level, lowered SOD level, and up-regulated mRNA and protein levels of NLRP3, ASC, caspase-1, and p-NF-κB p65/NF-κB p65. Compared with the model group, the drug administration in all the groups reduced inflammatory cell infiltration in the lung tissue. The inhibitor group and the western drug group showed enlarged alveolar cavity, thinned interstitium, and reduced inflammation. There was a small amount of alveolar wall rupture in the high-and medium-dose GQYY groups and reduced inflammatory cell infiltration in the low dose GQYY group. Compared with the model group, drug administration lowered level of ROS in the left and right lungs, lowered the MDA level, elevated the SOD level, and down-regulated the mRNA and protein levels of NLRP3, ASC, caspase-1, and p-NF-κB p65/NF-κB p65. GQYY can effectively reduce the damage caused by radiation and bystander effect, which may be associated with the ROS-mediated NLRP3 inflammasome activation.
Rats ; Animals ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; NF-kappa B/metabolism* ; Inflammasomes/metabolism* ; Lung Injury/genetics* ; Reactive Oxygen Species/metabolism* ; Bystander Effect ; Ointments ; Rats, Wistar ; Lung/metabolism* ; Caspase 1/metabolism* ; RNA, Messenger ; Superoxide Dismutase