OBJECTIVE: To investigate the effects of asperuloside on cervical cancer based on endoplasmic reticulum (ER) stress and mitochondrial pathway. METHODS: Different doses (12.5-800 µg/mL) of asperuloside were used to treat cervical cancer cell lines Hela and CaSki to calculate the half maximal inhibitory concentration (IC₅₀) of asperuloside. The cell proliferation was analyzed by clone formation assay. Cell apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by flow cytometry. The protein expressions of cleaved-caspase-3, Bcl-2, Bax, Cyt-c, cleaved-caspase-4 and glucose-regulated protein 78 (GRP78) were analyzed by Western blot. And the inhibitor of ER stress, 4-phenyl butyric acid (4-PBA) was used to treat cervical cancer cells to further verify the role of ER stress in the apoptosis of cervical cancer cells induced by asperuloside. RESULTS: Asperuloside of 325, 650, and 1300 µg/mL significantly inhibited the proliferation and promoted apoptosis of Hela and CaSki cells (P<0.01). All doses of asperuloside significantly increased intracellular ROS levels, reduced mitochondrial membrane potential, significantly reduced Bcl-2 protein expression level, and increased Bax, Cyt-c, GRP78 and cleaved-caspase-4 expressions (P<0.01). In addition, 10 mmol/L 4-PBA treatment significantly promoted cell proliferation and reduced apoptosis (P<0.05), and 650 µg/mL asperuloside could reverse 4-PBA-induced increased cell proliferation, decreased apoptosis and cleaved-caspase-3, -4 and GRP78 protein expressions (P<0.05). CONCLUSION Our study revealed the role of asperuloside in cervical cancer, suggesting that asperuloside promotes apoptosis of cervical cancer cells through ER stress-mitochondrial pathway.
Female ; Humans ; Uterine Cervical Neoplasms/metabolism* ; Caspase 3/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Reactive Oxygen Species/metabolism* ; Endoplasmic Reticulum Chaperone BiP ; HeLa Cells ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Endoplasmic Reticulum Stress ; Cell Line, Tumor

OBJECTIVE: To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism. METHODS: B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR. RESULTS: In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells. CONCLUSIONS Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.
Mice ; Animals ; Alocasia/metabolism* ; MAP Kinase Signaling System ; Caspase 3/metabolism* ; Apoptosis ; RNA, Messenger/metabolism*

Objective: To explore the effect of lncRNA ADPGK-AS1 on the proliferation and apoptosis of retinoblastoma cells and its possible mechanism. Methods: The tumor tissues of 31 patients with retinoblastoma admitted to Henan Provincial Eye Hospital from February to June 2020 and their corresponding normal tissues adjacent to the cancer were collected. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p in retinoblastoma tissues and normal adjacent tissues were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Human retinal epithelial cell ARPE-19, human retinoblastoma cell Y-79 and WERI-Rb-1 were cultured in vitro. The expression levels of lncRNA ADPGK-AS1 and miR-200b-5p were detected by qRT-PCR. Y-79 cells were randomly divided into si-con group, si-lncRNA ADPGK-AS1 group, miR con group, miR-200b-5p group, si-lncRNA ADPGK-AS1+ anti-miR con group, and si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group. The proliferation, cloning and apoptosis of cells in each group were detected by tetramethylazol blue method, plate cloning test and flow cytometry, respectively. The targeting relationship between lncRNA ADPGK-AS1 and miR-200b-5p was detected by double luciferase report test, and the expression level of cleaved-caspase-3 protein was detected by western blot. Results: Compared with the adjacent tissues, the expression of lncRNA ADPGK-AS1 in retinoblastoma tissues was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with ARPE-19 cells, the expression of lncRNA ADPGK-AS1 in Y-79 and WERI-Rb-1 cells was increased (P<0.05), while the expression of miR-200b-5p was decreased (P<0.05). Compared with the si-con group, the cell viability of the si-lncRNA ADPGK-AS1 group was reduced (1.06±0.09 vs 0.53±0.05, P<0.05), the number of cell clone formation was reduced (114.00±8.03 vs 57.00±4.13, P<0.05), while the apoptosis rate [(7.93±0.68)% vs (25.43±1.94)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). Compared with the miR-con group, the cell viability of the miR-200b-5p group was decreased (1.05±0.08 vs 0.57±0.05, P<0.05), the number of cell clone formation was decreased (118.00±10.02 vs 64.00±5.13, P<0.05), while the apoptosis rate [(7.89±0.71)% vs (23.15±1.62)%] and the protein level of cleaved-caspase-3 were increased (P<0.05). lncRNA ADPGK-AS1 could target the expression of miR-200b-5p. Compared with the si-lncRNA ADPGK-AS1+ anti-miR-con group, cell viability of the si-lncRNA ADPGK-AS1+ anti-miR-200b-5p group was increased (0.53±0.04 vs 1.25±0.10, P<0.05), and the number of cell clones was increased (54.00±4.39 vs 125.00±10.03, P<0.05), while the rate of apoptosis [(25.38±1.53)% vs (9.76±0.71)%] and the protein level of cleaved-caspase-3 were decreased (P<0.05). Conclusion: Interfering with the expression of lncRNA ADPGK-AS1 could inhibit the proliferation and clone formation and induce apoptosis of retinoblastoma cells by targeting the expression of miR-200b-5p.
Humans ; MicroRNAs/metabolism* ; Retinoblastoma/pathology* ; Caspase 3/metabolism* ; RNA, Long Noncoding/metabolism* ; Antagomirs/pharmacology* ; Cell Proliferation ; Cell Line, Tumor ; Apoptosis/genetics* ; Retinal Neoplasms/genetics* ; Gene Expression Regulation, Neoplastic ; Cell Movement/genetics*

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.
Animals ; Male ; Rats ; Apoptosis ; Brain Ischemia/metabolism* ; Caspase 3 ; Interleukin-1 ; Interleukin-6 ; Rats, Sprague-Dawley ; Reperfusion Injury/metabolism* ; Tumor Necrosis Factor-alpha/genetics* ; Flavonoids/pharmacology* ; Rhododendron/chemistry*

Based on network pharmacology, molecular docking, and in vitro experimental verification, this study aims to explore the effect of Albiziae Cortex-Tribuli Fructus combination on HSC-LX2 pyroptosis. Specifically, the targets of Albiziae Cortex, Tribuli Fructus, and hepatic fibrosis were retrieved from an online database and CNKI, and "drug-component-target" network and "drug-component-target-disease" network were constructed. Protein-protein interaction(PPI) network was established based on STRING. Metascape was employed for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and the mechanism of Albiziae Cortex-Tribuli Fructus combination against liver fibrosis was predicted. Molecular docking was used to verify some of the results of network pharmacology, and in vitro experiment was carried out to further verify the above conclusions. According to the results of network pharmacological analysis, 25 active components and 439 targets of Albiziae Cortex-Tribuli Fructus combination and 152 anti-liver fibrosis targets were screened out, including nucleotide-binding oligomerization domain and leucine-rich-repeat-and pyrin-domain-containing 3(NLRP3) and caspase-1. The key targets were involved in 194 KEGG pathways in which the NOD-like receptor signaling pathway topped. The binding common targets were related to pyroptosis. The results of in vitro experiment showed that the pair-containing serum reduced the proliferation rate of HSC-LX2 and the content of reactive oxygen species(ROS), interleukin-18(IL-18), and interleukin-1β(IL-1β)(P<0.05). Western blot and qRT-PCR suggested that the protein and gene expression of NLRP3, caspase-1, α-smooth muscle actin(α-SMA), and gasdermin D(GSDMD) in HSC-LX2 increased after AngⅡ stimulation, and the expression decreased after the intervention of pair-containing serum(P<0.05). In summary, the pair-containing serum can inhibit the classic pathway of pyroptosis, which may be the anti-liver fibrosis mechanism. This is consistent with the predicted results of network pharmacology.
Humans ; Hepatic Stellate Cells ; Network Pharmacology ; Molecular Docking Simulation ; NLR Family, Pyrin Domain-Containing 3 Protein ; Caspase 1/genetics* ; Fibrosis ; Drugs, Chinese Herbal/pharmacology*

The aim of this study was to explore the effects of Huangqin Tang(HQT) on the NLRP3/Caspase-1 signaling pathway in mice with DSS-induced ulcerative colitis(UC). C57BL/6J mice were randomly divided into a blank group, a model group(DSS group), and low-, medium-and high-dose HQT groups(HQT-L, HQT-M, and HQT-H), and western medicine mesalazine group(western medicine group). The UC model was induced in mice. Subsequently, the mice in the HQT-L, HQT-M, HQT-H groups, and the western medicine group were given low-, medium-, high-dose HQT, and mesalazine suspension by gavage, respectively, while those in the blank and DSS groups were given an equal volume of distilled water by gavage. After 10 days of administration, the body weight, DAI scores, and colonic histopathological score of mice in each group were determined. The levels of IL-6, IL-10, IL-1β, and TNF-α in serum were determined by ELISA. The mRNA expression of NLRP3 and Caspase-1 in colon tissues was determined by RT-qPCR. The protein expression of NLRP3 and Caspase-1 in colon tissues was detected by immunohistochemistry. The results showed that compared with the blank group, the DSS group showed decreased body weight of mice and increased DAI scores and intestinal histopathological score. Compared with the DSS group, the HQT groups and the western medicine group showed improved DAI scores, especially in the HQT-M, HQT-H, and the western medicine groups(P<0.05). The intestinal histopathological scores of the HQT groups and the western medicine group significantly decreased, especially in the HQT-M, HQT-H, and the western medicine groups(P<0.05). In addition, compared with the blank group, the DSS group showed elevated expression of NLRP3 and Caspase-1 in colon tissues, increased serum levels of IL-6, IL-1β, and TNF-α, and decreased IL-10 level. Compared with the DSS group, the HQT groups and the western medicine group displayed decreased expression of NLRP3 and Caspase-1 in colon tissues, reduced serum levels of IL-6, IL-1β, and TNF-α, and increased IL-10 level. The improvement was the most significant in the HQT-H group and the western medicine group(P<0.01). In conclusion, HQT may reduce the expression of NLRP3 and Caspase-1 in colon tissues, reduce the se-rum levels of IL-6, IL-1β, and TNF-α, and increase the expression of IL-10 by regulating the classic pyroptosis pathway of NLRP3/Caspase-1, thereby improving the symptoms of intestinal injury and inflammatory infiltration of intestinal mucosa in DSS mice to achieve its therapeutic effect.
Animals ; Mice ; Caspase 1/genetics* ; Colitis, Ulcerative/genetics* ; Colon ; Dextran Sulfate/adverse effects* ; Disease Models, Animal ; Interleukin-10/genetics* ; Interleukin-6/genetics* ; Mesalamine/pharmacology* ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Scutellaria baicalensis/chemistry* ; Tumor Necrosis Factor-alpha/metabolism* ; Drugs, Chinese Herbal/pharmacology*

This study aimed to parallelly investigate the cardioprotective activity of Cinnamomi Ramulus formula granules(CRFG) and Cinnamomi Cortex formula granules(CCFG) against acute myocardial ischemia/reperfusion injury(MI/RI) and the underlying mechanism based on the efficacy of "warming and coordinating the heart Yang". Ninety male SD rats were randomly divided into a sham group, a model group, CRFG low and high-dose(0.5 and 1.0 g·kg~(-1)) groups, and CCFG low and high-dose(0.5 and 1.0 g·kg~(-1)) groups, with 15 rats in each group. The sham group and the model group were given equal volumes of normal saline by gavage. Before modeling, the drug was given by gavage once a day for 7 consecutive days. One hour after the last administration, the MI/RI rat model was established by ligating the left anterior descending artery(LAD) for 30 min ischemia followed by 2 h reperfusion except the sham group. The sham group underwent the same procedures without LAD ligation. Heart function, cardiac infarct size, cardiac patho-logy, cardiomyocyte apoptosis, cardiac injury enzymes, and inflammatory cytokines were determined to assess the protective effects of CRFG and CCFG against MI/RI. The gene expression levels of nucleotide-binding oligomerization domain-like receptor family pyrin domain protein 3(NLRP3) inflammasome, apoptosis-associated speck-like protein containing a CARD(ASC), cysteinyl aspartate specific proteinase-1(caspase-1), Gasdermin-D(GSDMD), interleukin-1β(IL-1β), and interleukin-18(IL-18) were determined by real-time quantitative polymerase chain reaction(RT-PCR). The protein expression levels of NLRP3, caspase-1, GSDMD, and N-GSDMD were determined by Western blot. The results showed that both CRFG and CCFG pretreatments significantly improved cardiac function, decreased the cardiac infarct size, inhibited cardiomyocyte apoptosis, and reduced the content of lactic dehydrogenase(LDH), creatine kinase MB isoenzyme(CK-MB), aspartate transaminase(AST), and cardiac troponin Ⅰ(cTnⅠ). In addition, CRFG and CCFG pretreatments significantly decreased the levels of IL-1β, IL-6, and tumor necrosis factor-α(TNF-α) in serum. RT-PCR results showed that CRFG and CCFG pretreatment down-regulated the mRNA expression levels of NLRP3, caspase-1, ASC, and downstream pyroptosis-related effector substances including GSDMD, IL-18, and IL-1β in cardiac tissues. Western blot revealed that CRFG and CCFG pretreatments significantly decreased the protein expression levels of NLRP3, caspase-1, GSDMD, and N-GSDMD in cardiac tissues. In conclusion, CRFG and CCFG pretreatments have obvious cardioprotective effects on MI/RI in rats, and the under-lying mechanism may be related to the inhibition of NLRP3/caspase-1/GSDMD signaling pathway to reduce the cardiac inflammatory response.
Male ; Animals ; Rats ; Rats, Sprague-Dawley ; Interleukin-18 ; Myocardial Reperfusion Injury ; NLR Family, Pyrin Domain-Containing 3 Protein ; Tumor Necrosis Factor-alpha ; Myocardial Infarction ; Caspase 1

This study aims to investigate the effect of Astragali Radix-Curcumae Rhizoma(AC) combination on the proliferation, migration, and invasion of colon cancer HT-29 cells based on epithelial-mesenchymal transition(EMT). HT-29 cells were respectively treated with 0, 3, 6 and 12 g·kg~(-1) AC-containing serum for 48 h. The survival and growth of cells were measured by thiazole blue(MTT) colorimetry, and the proliferation, migration, and invasion of cells were detected by 5-ethynyl-2'-deoxyuridine(EdU) test and Transwell assay. Cell apoptosis was examined by flow cytometry. The BALB/c nude mouse model of subcutaneous colon cancer xenograft was established, and then model mice were classified into blank control group, 6 g·kg~(-1) AC group, and 12 g·kg~(-1) AC group. The tumor weight and volume of mice were recorded, and the histopathological morphology of the tumor was observed based on hematoxylin-eosin(HE) staining. The expression of apoptosis-associated proteins B-cell lymphoma-2-associated X protein(Bax), cysteine-aspartic acid protease-3(caspase-3), and cleaved caspase-3, and EMT-associated proteins E-cadherin, MMP9, MMP2 and vimentin in HT-29 cells and mouse tumor tissues after the treatment of AC was determined by Western blot. The results showed that cell survival rate and the number of cells at proliferation stage decreased compared with those in the blank control group. The number of migrating and invading cells reduced and the number of apoptotic cells increased in the administration groups compared with those in the blank control group. As for the in vivo experiment, compared with the blank control group, the administration groups had small tumors with low mass and shrinkage of cells and karyopycnosis in the tumor tissue, indicating that the AC combination may improve EMT. In addition, the expression of Bcl2 and E-cadherin increased and the expression of Bax, caspase-3, cleaved caspase-3, MMP9, MMP2, and vimentin decreased in HT-29 cells and tumor tissues in each administration group. In summary, the AC combination can significantly inhibit the proliferation, invasion, migration, and EMT of HT-29 cells in vivo and in vitro and promote the apoptosis of colon cancer cells.
Humans ; Animals ; Mice ; Caspase 3 ; Matrix Metalloproteinase 2 ; Matrix Metalloproteinase 9 ; Vimentin ; HT29 Cells ; bcl-2-Associated X Protein ; Colonic Neoplasms ; Cell Proliferation

Objective: To investigate the potential function and related mechanism of microRNA-223 (miRNA-223) in the podocyte pyroptosis of hepatitis B virus (HBV)-associated glomerulonephritis induced by HBV X protein (HBx). Methods: HBx-overexpressing lentivirus was transfected into human renal podocytes to mimic the pathogenesis of HBV-GN. Real-time fluorescence quantitative PCR and Western blotting experiments were used to detect the mRNA and protein expression of pyroptosis-related proteins [nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1], and inflammatory factors (interleukin-1β and interleukin-18), respectively.TUNEL staining and flow cytometry were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression of podocytes biomarkers desmin and nephrin; Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei. Enzyme-linked immunosorbent assay was used to measure caspase-1 activity. The dual luciferase reporter gene assay was used to verify the downstream target of miRNA-223. Podocytes were divided into the following nine groups: control group (no special treatment), empty plasmid group (transfected with empty plasmid), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+miRNA-223 mimic group (transfected with HBx overexpression lentivirus and miRNA-223 mimic), HBx overexpression+miRNA-223 inhibitor group (transfected with HBx overexpression lentivirus and miRNA-223 inhibitor), HBx overexpression+miRNA-223 mimic+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 mimic+ NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 siRNA), HBx overexpression+miRNA-223 inhibitor+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 inhibitor+NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 siRNA). Results: miRNA-223 was down-regulated in HBx overexpression group compared with the control group (P < 0.05). TUNEL and immunofluorescence staining showed that NLRP3 knockdown attenuated podocyte injury and pyroptosis induced by HBx overexpression (P < 0.05). Dual luciferase reporter gene assay demonstrated that NLRP3 was one of the downstream targets of miRNA-223. Rescue experiments revealed that NLRP3 overexpression weakened the protective effect of miRNA-223 in podocyte injury (P < 0.05). The addition of miRNA-223 mimic and NLRP3 siRNA decreased the expression of NLRP3 inflammasome and cytokines, and reduced the number of pyroptosis cells induced by HBx overexpression (all P < 0.05); The addition of miRNA-223 inhibitor and NLRP3 overexpression plasmid significantly increased the expression of NLRP3 inflammasome and cytokines, caspase-1 activity, and the number of pyroptosis cells (all P < 0.05). Conclusion: HBx may promote podocyte pyroptosis of HBV-GN via downregulating miRNA-223 targeting NLRP3 inflammasome, suggesting that miRNA-223 is expected to be a potential target for the treatment of HBV-GN.
Humans ; Inflammasomes/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pyroptosis ; Podocytes/metabolism* ; Hepatitis B virus/genetics* ; Caspase 1/metabolism* ; Cytokines/metabolism* ; Carrier Proteins/metabolism* ; MicroRNAs/genetics* ; Glomerulonephritis/metabolism* ; RNA, Small Interfering

OBJECTIVES: To study the protective effect of breviscapine against brain injury induced by intrauterine inflammation in preterm rats and its mechanism. METHODS: A preterm rat model of brain injury caused by intrauterine inflammation was prepared by intraperitoneal injections of lipopolysaccharide in pregnant rats. The pregnant rats and preterm rats were respectively randomly divided into 5 groups: control, model, low-dose breviscapine (45 mg/kg), high-dose breviscapine (90 mg/kg), and high-dose breviscapine (90 mg/kg)+ML385 [a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor, 30 mg/kg] (n=10 each). The number and body weight of the live offspring rats were measured for each group. Hematoxylin-eosin staining was used to observe the pathological morphology of the uterus and placenta of pregnant rats and the pathological morphology of the brain tissue of offspring rats. Immunofluorescent staining was used to measure the co-expression of ionized calcium binding adaptor molecule-1 (IBA-1) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex of offspring rats. ELISA was used to measure the levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1β (IL-1β) in the brain tissue of offspring rats. Western blotting was used to measure the expression of Nrf2 pathway-related proteins in the brain tissue of offspring rats. RESULTS: Pathological injury was found in the uterus, and placenta tissue of the pregnant rats and the brain tissue of the offspring rats, and severe microglia pyroptosis occurred in the cerebral cortex of the offspring rats in the model group. Compared with the control group, the model group had significant reductions in the number and body weight of the live offspring rats and the protein expression levels of Nrf2 and heme oxygenase-1 (HO-1) in the brain tissue of the offspring rats (P<0.05), but significant increases in the relative fluorescence intensity of the co-expression of IBA-1 and NLRP3, the levels of the inflammatory factors IL-6, IL-8, and IL-1β, and the protein expression levels of NLRP3 and caspase-1 in the brain tissue of the offspring rats (P<0.05). Compared with the model group, the breviscapine administration groups showed alleviated pathological injury of the uterus and placenta tissue of the pregnant rats and the brain tissue of the offspring rats, significant increases in the number and body weight of the live offspring rats and the protein expression levels of Nrf2 and HO-1 in the brain tissue of the offspring rats (P<0.05), and significant reductions in the relative fluorescence intensity of the co-expression of IBA-1 and NLRP3, the levels of the inflammatory factors IL-6, IL-8, and IL-1β, and the protein expression levels of NLRP3 and caspase-1 in the brain tissue of the offspring rats (P<0.05). The high-dose breviscapine group had a significantly better effect than the low-dose breviscapine (P<0.05). ML385 significantly inhibited the intervention effect of high-dose breviscapine (P<0.05). CONCLUSIONS Breviscapine can inhibit inflammatory response in brain tissue of preterm rats caused by intrauterine inflammation by activating the Nrf2 pathway, and it can also inhibit microglial pyroptosis and alleviate brain injury.
Animals ; Female ; Pregnancy ; Rats ; Body Weight ; Brain Injuries/prevention & control* ; Caspase 1 ; Inflammation/drug therapy* ; Interleukin-6 ; Interleukin-8 ; NF-E2-Related Factor 2 ; NLR Family, Pyrin Domain-Containing 3 Protein ; Flavonoids/therapeutic use*