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*

Objective To investigate the effects of miR-181b-5p on cells proliferation and apoptosis in acute myeloid leukemia (AML) by targeting paired box 9 (PAX9). Methods The relationship between expression level of PAX9 and prognosis in AML patients was analyzed by gene expression profiling interactive analysis (GEPIA) database and The Cancer Genome Atlas (TCGA) database. Kasumi-1 and AML5 cells were transfected with empty vector (Vector group) or PAX9 (PAX9 group). The proliferation activity was detected by CCK-8 assay, and cells cycle and apoptosis were detected by flow cytometry. Expressions of cyclin-dependent kinase 2 (CDK2), cyclin B1 (CCNB1), B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (BAX) were detected by Western blot analysis. The targeted microRNA (miRNA) by PAX9 was predicted by bioinformatics analysis, and the targeted effect was verified by luciferase reporter assay. The level of PAX9 mRNA was detected by real-time quantitative PCR, and expression of PAX9 protein was detected by Western blot analysis. Kasumi-1 and AML5 cells were transfected with miR-NC (miR-NC group) or miR-181b-5p (miR-181b-5p group). The cells were further transfected with PAX9 (miR-181b-5p combined with PAX9 group) in miR-181b-5p group. The proliferation, cycle and apoptosis of cells were detected by the above methods.Results GEPIA and TCGA databases showed that the expression of PAX9 was down-regulated in AML patients, which was correlated with poor prognosis. In Kasumi-1 and AML5 cells, compared with Vector group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in PAX9 group. It was confirmed by double luciferase reporter assay that PAX9 was the target gene of miR-181b-5p. Compared with miR-NC group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were increased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were decreased in miR-181b-5p group. Compared with miR-181b-5p group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in miR-181b-5p combined with PAX9 group. Conclusion The miR-181b-5p can promote the proliferation of AML cells and delay apoptosis by inhibiting PAX9.
Humans ; Apoptosis/genetics* ; bcl-2-Associated X Protein ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Leukemia, Myeloid, Acute/pathology* ; Luciferases ; MicroRNAs/metabolism* ; PAX9 Transcription Factor/genetics*

OBJECTIVES: To explore the role and potential mechanisms of chitinase-3-like protein 1 (CHI3L1) in coronary artery lesions in a mouse model of Kawasaki disease (KD)-like vasculitis. METHODS: Four-week-old male SPF-grade C57BL/6 mice were randomly divided into a control group and a model group, with 10 mice in each group. The model group mice were intraperitoneally injected with 0.5 mL of lactobacillus casei cell wall extract (LCWE) to establish a mouse model of KD-like vasculitis, while the control group mice were injected with an equal volume of normal saline. The general conditions of the mice were observed on the 3rd, 7th, and 14th day after injection. Changes in coronary artery tissue pathology were observed using hematoxylin-eosin staining. The level of CHI3L1 in mouse serum was measured by enzyme-linked immunosorbent assay. Immunofluorescence staining was used to detect the expression and localization of CHI3L1, von Willebrand factor (vWF), and α-smooth muscle actin (α-SMA) in coronary artery tissue. Western blot analysis was used to detect the expression of CHI3L1, vWF, vascular endothelial cadherin (VE cadherin), Caspase-3, B cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), nuclear factor κB (NF-κB), and phosphorylated NF-κB (p-NF-κB) in coronary artery tissue. RESULTS: The serum level of CHI3L1 in the model group was significantly higher than that in the control group (P<0.05). Compared to the control group, the expression of CHI3L1 in the coronary artery tissue was higher, while the expression of vWF was lower in the model group. The relative expression levels of CHI3L1, Bax, Caspase-3, NF-κB, and p-NF-κB were significantly higher in the model group than in the control group (P<0.05). The relative expression levels of vWF, VE cadherin, and Bcl-2 were lower in the model group than in the control group (P<0.05). CONCLUSIONS In the LCWE-induced mouse model of KD-like vasculitis, the expression levels of CHI3L1 in serum and coronary arteries increase, and it may play a role in coronary artery lesions through endothelial cell apoptosis mediated by inflammatory reactions.
Male ; Animals ; Mice ; Mucocutaneous Lymph Node Syndrome/pathology* ; Coronary Vessels/pathology* ; NF-kappa B ; Caspase 3/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Chitinase-3-Like Protein 1 ; von Willebrand Factor/metabolism* ; Mice, Inbred C57BL ; Cadherins

To investigate the antidepressant mechanism of Shenling Kaixin Granules(SLKX) in treating chronic unpredictable mild stress(CUMS) model rats. Ninety male SD rats were randomly divided into control group, model group, Shugan Jieyu Capsules(110 mg·kg~(-1)) group and SLKX low-(90 mg·kg~(-1)), medium-(180 mg·kg~(-1)), and high-dose(360 mg·kg~(-1)) groups. Depression rat model was replicated by CUMS method. After treatment, the behavioral changes of rats were evaluated by sugar preference, open field, elevated cross maze and forced swimming experiments. The contents of interleukin 1 beta(IL-1β), tumor necrosis factor α(TNF-α), brain-derived neurotrophic factor(BDNF) and 5-hydroxytryptamine(5-HT) in serum were determined by enzyme linked immunosorbent assay(ELISA), and the activities of superoxide dismutase(SOD) and catalase(CAT) in hippocampal CA1 region were also detected. Pathological changes in hippocampal CA1 region were detected by hematoxylin-eosin(HE) staining, and Western blot was used to determine the expression of nerve growth factor(NGF), BDNF, phospho-tyrosine kinase receptor(p-TrkB)/TrkB, phospho-cAMP-response element binding protein(p-CREB)/CREB, nuclear factor E2 related factor 2(Nrf2), heme oxygenase 1(HO-1), B-cell lymphoma-2(Bcl-2)/Bcl-2 associated X protein(Bax) and caspase-3 in hippocampal CA1 region. RESULTS:: showed that compared with the control group, the model group had decreased sugar preference, reduced number of entries and time spent in the center of open field and shortened total distance of movement, reduced number of entries and proportion of time spent in open arm, and increased number and time of immobility in forced swimming experiment. Additionally, the serum contents of IL-1β and TNF-α and the expression of caspase-3 were higher, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1 and Bcl-2/Bax, and the Nrf2 nuclear translocation were lower in model group than in control group. Compared with the conditions in model group, the sugar preference, the number of entries and time spent in the center of open, total distance of movement, and the number of entries and proportion of time spent in open arm in treatment groups were increased while the number and time of immobility in forced swimming experiment were decreased; the serum contents of IL-1β and TNF-α and the expression of caspase-3 were down regulated, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1, Bcl-2/Bax, and Nrf2 nuclear translocation were enhanced. In conclusion, SLKX might regulate the Nrf2 nucleus translocation by activating BDNF/TrkB/CREB pathway, lower oxidative stress damage in hippocampus, inhibit caspase-3 activity, and reduce apoptosis of hippocampal nerve cells, thereby playing an antidepressant role.
Rats ; Male ; Animals ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Nerve Growth Factor/metabolism* ; Brain-Derived Neurotrophic Factor/metabolism* ; Signal Transduction ; Tumor Necrosis Factor-alpha/metabolism* ; Serotonin/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Rats, Sprague-Dawley ; Antidepressive Agents/pharmacology* ; Hippocampus/metabolism* ; Superoxide Dismutase/metabolism* ; Sugars/pharmacology* ; Depression/genetics* ; Stress, Psychological/metabolism*

This study aims to investigate the therapeutic effect of alcohol extract of root and root bark of Toddalia asiatica(TAAE) on collagen-induced arthritis(CIA) in rats through phosphatidylinoinosidine-3 kinase/protein kinase B(PI3K/Akt) signaling pathway. To be specific, CIA was induced in rats, and then the rats were treated(oral, daily) with TAAE and Tripterygium Glycoside Tablets(TGT), respectively. The swelling degree of the hind leg joints was scored weekly. After 35 days of administration, the histopathological changes were observed based on hematoxylin and eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the levels of cytokines [tumor necrosis factor-α(TNF-α), interleukin(IL)-6)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining was performed to detect the apoptosis of synoviocytes in rats. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma 2(Bcl-2)-associated X(Bax), Bcl-2, and caspase-3 and pathway-related proteins phosphoinositide 3-kinase(PI3K), phosphorylated(p)-PI3K, protein kinase B(Akt), and p-Akt. RT-qPCR was conducted to examine the mRNA levels of Bax, Bcl-2, caspase-3, TNF-α, IL-6, and IL-1β and pathway-related proteins PI3K, p-PI3K, Akt, and p-Akt. TAAE can alleviate the joint swelling in CIA rats, reduce serum levels of inflammatory cytokines, improve synovial histopathological changes, promote apoptosis of synoviocytes, and inhibit synovial inflammation. In addition, RT-qPCR and Western blot results showed that TAAE up-regulated the level of Bax, down-regulated the level of Bcl-2, and activated caspase-3 to promote apoptosis in synoviocytes. TAAE effectively down-regulated the protein levels of p-PI3K and p-Akt. In this study, TAAE shows therapeutic effect on CIA in rats and reduces the inflammation. The mechanism is that it suppresses PI3K/Akt signaling pathway and promotes synoviocyte apoptosis. Overall, this study provides a new clue for the research on the anti-inflammatory mechanism of TAAE and lays a theoretical basis for the better clinical application of TAAE in the treatment of inflammatory and autoimmune diseases.
Rats ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Caspase 3/genetics* ; Tumor Necrosis Factor-alpha/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Plant Bark ; Anti-Inflammatory Agents/therapeutic use* ; Arthritis, Experimental/chemically induced* ; Inflammation/drug therapy* ; Cytokines/metabolism* ; Proto-Oncogene Proteins c-bcl-2 ; Apoptosis

This study aims to explore the neuroprotective mechanism of ginsenoside Re(GS-Re) on drosophila model of Parkinson's disease(PD) induced by rotenone(Rot). To be specific, Rot was used to induce PD in drosophilas. Then the drosophilas were grouped and respectively treated(GS-Re: 0.1, 0.4, 1.6 mmol·L~(-1); L-dopa: 80 μmol·L~(-1)). Life span and crawling ability of drosophilas were determined. The brain antioxidant activity [content of catalase(CAT), malondialdehyde(MDA), reactive oxygen species(ROS), superoxide dismutase(SOD)], dopamine(DA) content, and mitochondrial function [content of adenosine triphosphate(ATP), NADH:ubiquinone oxidoreductase subunit B8(NDUFB8) Ⅰ activity, succinate dehydrogenase complex, subunit B(SDHB) Ⅱ activity] were detected by enzyme-linked immunosorbent assay(ELISA). The number of DA neurons in the brains of drosophilas was measured with the immunofluorescence method. The levels of NDUFB8 Ⅰ, SDHB Ⅱ, cytochrome C(Cyt C), nuclear factor-E2-related factor 2(Nrf2), heme oxygenase-1(HO-1), B-cell lymphoma/leukemia 2(Bcl-2)/Bcl-2-assaciated X protein(Bax), and cleaved caspase-3/caspase-3 in the brain were detected by Western blot. The results showed that model group [475 μmol·L~(-1) Rot(IC_(50))] demonstrated significantly low survival rate, obvious dyskinesia, small number of neurons and low DA content in the brain, high ROS level and MDA content, low content of SOD and CAT, significantly low ATP content, NDUFB8 Ⅰ activity, and SDHB Ⅱ activity, significantly low expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax, large amount of Cyt C released from mitochondria to cytoplasm, low nuclear transfer of Nrf2, and significantly high expression of cleaved caspase-3/caspase-3 compared with the control group. GS-Re(0.1, 0.4, and 1.6 mmol·L~(-1)) significantly improved the survival rate of PD drosophilas, alleviated the dyskinesia, increased DA content, reduced the loss of DA neurons, ROS level, and MDA content in brain, improved content of SOD and CAT and antioxidant activity in brain, maintained mitochondrial homeostasis(significantly increased ATP content and activity of NDUFB8 Ⅰ and SDHB Ⅱ, significantly up-regulated expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax), significantly reduced the expression of Cyt C, increased the nuclear transfer of Nrf2, and down-regulated the expression of cleaved caspase-3/caspase-3. In conclusion, GS-Re can significantly relieve the Rot-induced cerebral neurotoxicity in drosophilas. The mechanism may be that GS-Re activates Keap1-Nrf2-ARE signaling pathway by maintaining mitochondrial homeostasis, improves antioxidant capacity of brain neurons, then inhibits mitochondria-mediated caspase-3 signaling pathway, and the apoptosis of neuronal cells, thereby exerting the neuroprotective effect.
Animals ; Reactive Oxygen Species/metabolism* ; Antioxidants/pharmacology* ; Oxidative Stress ; NF-E2-Related Factor 2/metabolism* ; Caspase 3/metabolism* ; Parkinson Disease/genetics* ; bcl-2-Associated X Protein/metabolism* ; Neuroprotective Agents/pharmacology* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Drosophila/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Superoxide Dismutase/metabolism* ; Adenosine Triphosphate/pharmacology*

The purpose of this study was to explore the effect and mechanism of Xihuang Pills on rats with precancerous lesions of the breast. Of 48 healthy female rats, 8 were randomly selected as blank group, and the other 40 were treated with 7,12-dimethylbenzanthracene(DMBA) combined with estrogen and progestin to establish a model of precancerous lesions of the breast. The successfully modeled rats were randomly divided into a model group, a tamoxifen group(1.8 mg·kg~(-1)·d~(-1)), a Xihuang Pills low-dose group(0.3 g·kg~(-1)·d~(-1)), a medium-dose group(0.6 g·kg~(-1)·d~(-1)) and a high-dose group(1.2 g·kg~(-1)·d~(-1)). After 30 days of admi-nistration, the histopathological changes of viscera and breast were observed by haematoxylin and eosin(HE) staining, and the visceral index was calculated. Enzyme linked immunosorbent assay(ELISA) was used to detect the contents of estradiol(E_2) and progesterone(P) in serum. The protein expressions of vascular endothelial growth factor(VEGF) and fibroblast growth factor 2(FGF2) were detected by immunohistochemistry. The protein expressions of VEGF, vascular endothelial growth factor receptor 2(VEGFR2), phosphorylated-vascular endothelial growth factor receptor 2(p-VEGFR2), B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were detected by Western blot and the mRNA expressions of VEGF, FGF2, CXC-chemokine receptor 4(CXCR4), cysteine aspartic acid-specific protease(caspase-3), and stromal cell-derived factor 1(SDF-1) were detected by real-time polymerase chain reaction(RT-PCR). HE staining revealed that the model group had some liver and kidney damages and severe hyperplastic mammary tissue, while the Xihuang Pills high-dose group had mild hyperplasia. Compared with the model group, the Xihuang Pills groups had lo-wer ovarian coefficient(P<0.05 or P<0.01) and Xihuang Pills high-dose group had lower uterine coefficient(P<0.01). ELISA results showed that compared with the model group, expressions of E_2 and P in Xihuang Pills high-dose group were significantly decreased(P<0.05 or P<0.01). Immunohistochemistry, Western blot and RT-PCR indicated that compared with the conditions in the model group, the protein and mRNA expressions of VEGF and FGF2 in the Xihuang Pills groups were down-regulated(P<0.05 or P<0.01), and the protein expression of Bcl-2 was lowered(P<0.01); there was a decrease in the protein expressions of VEGFR2 and p-VEGFR2(P<0.01), a down-regulation in the mRNA expressions of CXCR4 and SDF-1(P<0.01), while an increase in the mRNA expression of caspase-3(P<0.01) in both Xihuang Pills medium-dose and high-dose groups; the protein expression of Bax in Xihuang Pills high-dose group was increased(P<0.01). The above results indicated that Xihuang Pills can effectively intervene in precance-rous lesions of the breast, and the mechanism may be related to the regulation of E_2 and P secretion as well as the inhibition of angiogenesis and chemokine receptor expression, thus controlling the occurrence of precancerous lesions of the breast in rats.
Rats ; Female ; Animals ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; Vascular Endothelial Growth Factor A/metabolism* ; Caspase 3 ; Vascular Endothelial Growth Factor Receptor-2 ; Fibroblast Growth Factor 2 ; Proto-Oncogene Proteins c-bcl-2 ; 9,10-Dimethyl-1,2-benzanthracene/toxicity* ; Precancerous Conditions ; Hyperplasia ; Receptors, Chemokine ; RNA, Messenger

Objective To investigate the effects of formononetin (FMN) on cognitive behavior and inflammation in aging rats with chronic unpredictable mild stress (CUMS). Methods SD rats aged about 70 weeks were divided into healthy control group, CUMS model group, CUMS combined with 10 mg/kg FMN group, CUMS combined with 20 mg/kg FMN group and CUMS combined with 1.8 mg/kg fluoxetine hydrochloride (Flu) group. Except for healthy control group, other groups were stimulated with CUMS and administered drugs for 28 days. Sugar water preference, forced swimming experiment and open field experiment were used to observe the emotional behavior of rats in each group. HE staining was used to observe the pathological injury degree of brain equine area. The contents of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were detected by the kit. The apoptosis was tested by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) in the brain tissue. The levels of tumor necrosis factor α (TNF-α), inducible nitric oxide synthase (iNOS) and interleukin 6 (IL-6) in peripheral blood were measured by ELISA. Western blot analysis was used to detect Bcl2, Bcl2 associated X protein (BAX), cleaved caspase-9, cleaved caspase-3, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and phosphorylated nuclear factor κB p65 (p-NF-κB p65) in brain tissues. Results Compared with CUMS model group, sugar water consumption, open field activity time, open field travel distance and swimming activity time significantly increased in the CUMS combined with 20 mg/kg FMN group and the CUMS combined with 1.8 mg/kg Flu group. The number of new outarm entry increased significantly, while the number of initial arm entry and other arm entry decreased significantly. The pathological damage of brain equine area was alleviated, and the contents of 5-HT and 5-HIAA were significantly increased. The ratio of BAX/Bcl2 and the expression of cleaved caspase-9 and cleaved caspase-3 protein as well as the number of apoptotic cells were significantly decreased. The contents of TNF-α, iNOS and IL-6 were significantly decreased. The protein levels of TLR4, MyD88 and p-NF-κB p65 were significantly decreased. Conclusion FMN can inhibit the release of inflammatory factors by blocking NF-κB pathway and improve cognitive and behavioral ability of CUMS aged rats.
Rats ; Animals ; Horses ; NF-kappa B/metabolism* ; Signal Transduction ; bcl-2-Associated X Protein/metabolism* ; Toll-Like Receptor 4/metabolism* ; Caspase 3/metabolism* ; Caspase 9/metabolism* ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Myeloid Differentiation Factor 88 ; Hydroxyindoleacetic Acid/pharmacology* ; Serotonin/metabolism* ; Rats, Sprague-Dawley ; Hippocampus/metabolism* ; Cognition

OBJECTIVES: During pregnancy, pregnant women are prone to stress reactions due to external stimuli, affecting their own health and fetal development. At present, there is no good treatment for the stress reactions from pregnant women during pregnancy. This study aims to explore the effect of probiotics on abnormal behavior and hippocampal injury in pregnant stressed offspring. METHODS: SD pregnant rats were divided into a control group, a stress group, and a probiotics group, with 6 rats in each group. The control group was untreated; the stress group was given restraint stress on the 15th-20th day of pregnancy; the probiotics group was given both bifidobacterium trisporus capsules and restraint stress on the 15th-20th day of pregnancy, and the offspring continued to be fed with probiotics until 60 days after birth (P60). The offspring rats completed behavioral tests such as the open field test, the elevated plus maze test, the new object recognition test, and the barnes maze test at 60-70 d postnatally. Nissl's staining was used to reflect the injury of hippocampal neurons; immunohistochemical staining was used to detect the expression of microglia marker ionized calcium binding adapter molecule 1 (IBA-1) which can reflect microglia activation; ELISA was used to detect the content of plasma TNF-α and IL-1β; Western blotting was used to detect the expression of Bax, Bcl-2, and caspase-3. RESULTS: The retention time of offspring rats in the stress group in the central area of the open field was significantly less than that in the control group (P<0.01), and the retention time of offspring rats in the probiotic group in the central area of the open field was significantly more than that in the stress group (P<0.05). The offspring rats in the stress group stayed in the open arm for a shorter time than the control group (P<0.05) and entered the open arm less often than the control group (P<0.01); the offspring rats in the probiotic group stayed in the open arm for a longer time than the stress group and entered the open arm more often than the stress group (both P<0.05). The discrimination ratio for new to old objects in the offspring rats of the stress group was significantly lower than that of the control group (P<0.01), and the discrimination ratio for new to old objects in the offspring rats of the probiotic group was significantly higher than that of the stress group (P<0.05). The offspring rats in the stress group made significantly more mistakes than the control group (P<0.05), and the offspring rats in the probiotic group made significantly fewer mistakes than the stress group (P<0.05). Compared with the control group, the numbers of Nissl bodies in CA1, CA3, and DG area were significantly reduced in the offspring rats of the stress group (all P<0.001), the number of activated microglia in DG area of hippocampus was significantly increased (P<0.01), the contents of TNF-α and IL-1β in peripheral blood were significantly increased (P<0.05 or P<0.01), the protein expression level of Bcl-2 was significantly down-regulated, and the protein expression levels of Bax and caspase-3 were significantly up-regulated (all P<0.001). Compared with the stress group, the numbers of Nissl bodies in CA1, CA3, and DG area were significantly increased in the probiotic group offspring rats (P<0.001, P<0.01, P<0.05), the number of activated microglia in the DG area of hippocampus was significantly reduced (P<0.05), and the TNF-α and IL-1β levels in peripheral blood were significantly decreased (both P<0.05), the protein expression level of Bcl-2 was significantly up-regulated, and the protein expression levels of Bax and caspase-3 were significantly down-regulated (all P<0.001). CONCLUSIONS Probiotic intervention partially ameliorated anxiety and cognitive impairment in rats offspring of pregnancy stress, and the mechanism may be related to increasing the number of neurons, inhibiting the activation of hippocampal microglia, and reducing inflammation and apoptosis.
Animals ; Caspase 3/metabolism* ; Female ; Hippocampus/physiopathology* ; Humans ; Pregnancy ; Probiotics/therapeutic use* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Rats ; Stress, Psychological/therapy* ; Tumor Necrosis Factor-alpha/metabolism* ; bcl-2-Associated X Protein/metabolism*

Objective: To study the effects of dihydromyricetin (DMY) on the proliferation, apoptosis and epithelial mesenchymal transition (EMT) of esophageal squamous cell carcinoma (ESCC) cell KYSE150 and KYSE410. Methods: KYSE150 and KYSE410 cells were treated with different concentrations of DMY (0, 25, 50, 100, 150, 200 μmol/L) for 24 hours. The median inhibition concentration (IC₅₀) values of KYSE150 and KYSE410 were detected by cell counting kit-8 (CCK-8) method. Then 0.5‰ dimethyl sulfoxide (DMSO) was used as control group, dihydromyricetin (DMY), dihydromyricetin and transforming growth factor-β1 (DMY+ TGF-β1), transforming growth factor-β1 (TGF-β1) were used as experimental group. Cell proliferation and apoptosis rates were measured by clonal formation and flow cytometry. Transwell invasion and wound healing assay were used to detect cell invasion and migration. The protein expression levels of Caspase-3, Caspase-9, Bcl-2, Bax, Smad2/3, phosphorylation-Smad2/3 (p-Smad2/3) and Vimentin were detected by western blot. Results: The IC₅₀ values of DMY on KYSE410 and KYSE150 cells were 100.51 and 101.27 μmol/L. The clone formation numbers of KYSE150 and KYSE410 in DMY group [(0.53±0.03) and (0.31±0.03)] were lower than those in DMSO group [(1.00±0.10) and (1.00±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in DMY group [(1.84±0.22)% and (2.80±0.07)%] were higher than those in DMSO group [(1.00±0.18)% and (1.00±0.07)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in DMY group [(0.42±0.03) and (0.29±0.05)] were lower than those in DMSO group [(1.00±0.08) and (1.00±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in DMY group [(0.65±0.14)% and (0.40±0.17)%] were lower than those in DMSO group [(1.00±0.10)% and (1.00±0.08)%, P<0.05]. The clone formation numbers of KYSE150 and KYSE410 in TGF-β1 group [(1.01±0.08) and (0.99±0.25)] were higher than those in DMY+ TGF-β1 group [(0.73±0.10) and (0.58±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in TGF-β1 group [(0.81±0.14)% and (1.18±0.10)%] were lower than those in DMY+ TGF-β1 group [(1.38±0.22)% and (1.85±0.04)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in TGF-β1 group [(1.19±0.11) and (1.39±0.11)] were higher than those in DMY+ TGF-β1 group [(0.93±0.09) and (0.93±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in TGF-β1 group [(1.87±0.19)% and (1.32±0.04)%] were higher than those in DMY+ TGF-β1 group [(0.86±0.16)% and (0.77±0.12)%, P<0.05]. The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY group were higher than those in DMSO group, while the protein expression level of Bcl-2 was lower than that in DMSO group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in DMY group were lower than those in DMSO group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in TGF-β1 group were lower than those in DMY+ TGF-β1 group, and the protein expression level of Bcl-2 was higher than that in DMY+ TGF-β1 group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY+ TGF-β1 group were lower than those in DMY group, and the protein expression level of Bcl-2 was higher than that in DMY group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in TGF-β1 group were higher than those in DMY+ TGF-β1 group (P<0.05). Conclusion: DMY can inhibit the proliferation and EMT of ESCC mediated by TGF-β1 and promote cell apoptosis.
Apoptosis ; Caspase 3/metabolism* ; Caspase 9/metabolism* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Dimethyl Sulfoxide/pharmacology* ; Epithelial-Mesenchymal Transition ; Esophageal Neoplasms/metabolism* ; Esophageal Squamous Cell Carcinoma ; Flavonols ; Humans ; Signal Transduction ; Transforming Growth Factor beta1/pharmacology* ; Vimentin/metabolism* ; bcl-2-Associated X Protein/pharmacology*