OBJECTIVE: To investigate the effect of baicalin on the growth of extranodal NK/T cell lymphoma (ENKTCL) cells and its related mechanism. METHODS: Normal NK cells and human ENKTCL cells lines SNK-6 and YTS were cultured, then SNK-6 and YTS cells were treated with 5, 10, 20 μmol/L baicalin and set control. Cell proliferation and apoptosis was detected by Edu method and FCM method, respectively, and expressions of BCL-2, Bax, FOXO3 and CCL22 proteins were detected by Western blot. Interference plasmids were designed and synthesized. FOXO3 siRNA interference plasmids and CCL22 pcDNA overexpression plasmids were transfected with PEI transfection reagent. Furthermore, animal models were established for validation. RESULTS: In control group and 5, 10, 20 μmol/L baicalin group, the proliferation rate of SNK-6 cells was (56.17±2.96)%, (51.92±4.63)%, (36.42±1.58)%, and (14.60±2.81)%, respectively, while that of YTS cells was (58.85±2.98)%, (51.38±1.32)%, (34.75±1.09)%, and (15.45±1.10)%, respectively. In control group and 5, 10, 20 μmol/L baicalin group, the apoptosis rate of SNK-6 cells was (5.93±0.74)%, (11.78±0.34)%, (28.46±0.44)%, and (32.40±0.37)%, respectively, while that of YTS cells was (7.93±0.69)%, (16.29±1.35)%, (33.91±1.56)%, and (36.27±1.06)%, respectively. Compared with control group, the expression of BCL-2 protein both in SNK-6 and YTS cells decreased significantly (P<0.001), and the expression of Bax protein increased in SNK-6 cells only when the concentration of baicalin was 20 μmol/L (P<0.001), while that in YTS cells increased in all three concentrations(5, 10, 20 μmol/L) of baicalin (P<0.001). The expression of FOXO3 protein decreased while CCL22 protein increased in ENKTCL cell lines compared with human NK cells (P<0.001), but the expression of FOXO3 protein increased (P<0.01) and CCL22 protein decreased after baicalin treatment (P<0.001). Animal experiments showed that baicalin treatment could inhibit tumor growth. The expression of CCL22 protein in ENKTCL tissue of nude mice treated with baicalin decreased compared with control group (P<0.01), while the FOXO3 protein increased (P<0.05). In addition, FOXO3 silencing resulted in the decrease of FOXO3 protein expression and increase of CCL22 protein expression (P<0.01, P<0.001). CONCLUSION Baicalin can inhibit proliferation and promote apoptosis of ENKTCL cell lines SNK-6 and YTS, up-regulate the expression of Bax protein, down-regulate the expression of BCL-2 protein, and down-regulate the expression of CCL22 protein mediated by FOXO3. Animal experiment shown that the baicalin can inhibit tumor growth. Baicalin can inhibit the growth and induce apoptosis of ENKTCL cells through FOXO3/CCL22 signaling pathway.
Animals ; Mice ; Humans ; Lymphoma, Extranodal NK-T-Cell/pathology* ; Forkhead Box Protein O3/metabolism* ; bcl-2-Associated X Protein/pharmacology* ; Mice, Nude ; Signal Transduction ; Apoptosis ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Chemokine CCL22/pharmacology*

OBJECTIVE: To predict the targets and pathways in the therapeutic mechanism of Guizhi Gancao Decoction (GZGCD) against heart failure (HF) based on network pharmacology. METHODS: The chemical components of GZGCD were analyzed using the databases including TCMSP, TCMID and TCM@Taiwan, and the potential targets of GZGCD were predicted using the SwissTargetPrediction database. The targets of HF were obtained using the databases including DisGeNET, Drugbank and TTD. The intersection targets of GZGCD and HF were identified using VENNY. Uniport database was used to convert the information, and the components-targets-disease network was constructed using Cytoscape software. The Bisogene plug-in, Merge plug-in, and CytoNCA plug-in in Cytoscape software were used for protein-protein interaction (PPI) analysis to acquire the core targets. Metascape database was used for GO and KEGG analysis. The results of network pharmacology analysis were verified with Western blot analysis. Three factors (PKCα, ERK1/2 and BCL2) were screened according to the degree value of network pharmacology results and the degree of correlation with heart failure process. The pentobarbtal sodium was dissolvein H9C2 cells treated with serum-free high glucose medium to simulate the ischemic anoxic environment of heart failure. The total proteins of myocardial cells were extracted. The protein contents of PKCα, ERK1/2 and BCL2 were determined. RESULTS: We identified a total of 190 intersection targets between GZGCD and HF using Venny database, involving mainly the circulatory system process, cellular response to nitrogen compounds, cation homeostasis, and regulation of the MAPK cascade. These potential targets were also involved in 38 pathways, including the regulatory pathways in cancer, calcium signal pathway, cGMP-PKG signal pathway, and cAMP signal pathway. Western blot analysis showed that in an in vitro H9C2 cell model of HF, treatment with GZGCD downregulated PKCα and ERK1/2 expressions and upregulated BCL2 expression. CONCLUSION The therapeutic mechanism of GZGCD for HF involves multiple targets including PRKCA, PRKCB, MAPK1, MAPK3, and MAPK8 and multiple pathways including the regulatory pathway in cancer and the calcium signaling pathway.
Humans ; Protein Kinase C-alpha ; Network Pharmacology ; Heart Failure/drug therapy* ; Proto-Oncogene Proteins c-bcl-2

OBJECTIVE: To investigate the effect of phorbol-12-myristate-13-ace-tate (TPA) on the proliferation and apoptosis of acute promyelocytic leukemia cell line NB4 and its molecular mechanism. METHODS: The effect of different concentrations of TPA on the proliferation of NB4 cells at different time points was detected by CCK-8 assay. The morphological changes of NB4 cells were observed by Wright-Giemsa staining. The cell cycle and apoptosis of NB4 cells after TPA treatment were detected by flow cytometry. The mRNA expressions of NB4 cells after TPA treatment were analyzed by high-throughput microarray analysis and real-time quantitative PCR. Western blot was used to detect the protein expression of CDKN1A, CDKN1B, CCND1, MYC, Bax, Bcl-2, c-Caspase 3, c-Caspase 9, PIK3R6, AKT and p-AKT. RESULTS: Compared with the control group, TPA could inhibit the proliferation of NB4 cells, induce the cells to become mature granulocyte-monocyte differentiation, and also induce cell G₁ phase arrest and apoptosis. Differentially expressed mRNAs were significantly enriched in PI3K/AKT pathway. TPA treatment could increase the mRNA levels of CCND1, CCNA1, and CDKN1A, while decrease the mRNA level of MYC. It could also up-regulate the protein levels of CDKN1A, CDKN1B, CCND1, Bax, c-Caspase 3, c-Caspase 9, and PIK3R6, while down-regulate MYC, Bcl-2, and p-AKT in NB4 cells. CONCLUSION TPA induces NB4 cell cycle arrest in G₁ phase and promotes its apoptosis by regulating PIK3/AKT signaling pathway.
Humans ; Leukemia, Promyelocytic, Acute ; Caspase 3/metabolism* ; Caspase 9/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Cell Line, Tumor ; Cell Division ; Apoptosis ; RNA, Messenger ; Cell Proliferation

OBJECTIVE: To investigate the effect and underlying mechanism of hispidulin on the proliferation and apoptosis of leukemia K562 cells. METHODS: K562 cells were cultured in vitro and treated with 0, 5, 25 or 100 μmol/L hispidulin for 24 h. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. Western blot was used to assess the expression of Bax, Bcl-2 and interleukin (IL)-37 proteins. Bone marrow mononuclear cells were extracted from 17 chronic myeloid leukemia patients and 21 healthy individuals by Ficoll-Hypaque density gradient method, and the expression of IL-37 protein was measured by Western blot. K562 cells with IL-37 overexpression or knockdown were constructed, and then treated with 0 or 100 μmol/L hispidulin for 24 h. Cell proliferation, apoptosis and protein expression of Bax and Bcl-2 were determined in the same way as above. RESULTS: After K562 cells were treated with hispidulin, the cell inhibition rate, apoptosis rate, and the protein expression of Bax and IL-37 were significantly increased (P <0.05), but the cell proliferation and expression of Bcl-2 protein were decreased (P <0.05). The expression of IL-37 protein in bone marrow mononuclear cells of the leukemia patient was 0.24±0.03, which was significantly lower than 0.91±0.05 of healthy controls (P <0.05). Overexpression of IL-37 significantly promoted inhibition rate, apoptosis rate, and expression of Bax protein in K562 cells (P <0.05), but suppressed the expression of Bcl-2 protein (P <0.05). In addition, knockdown of IL-37 could reverse the effects of hispidulin on proliferation and apoptosis of K562 cells. CONCLUSION Hispidulin inhibits the proliferation and induces apoptosis of leukemia K562 cells, which may be related to the up-regulation of IL-37 protein in cells.
Humans ; K562 Cells ; bcl-2-Associated X Protein/pharmacology* ; Apoptosis ; Leukemia ; Proto-Oncogene Proteins c-bcl-2 ; Cell Proliferation

OBJECTIVE: To explore the mechanism of Radix Scrophulariae (RS) extracts in the treatment of hyperthyroidism rats by regulating proliferation, apoptosis, and autophagy of thyroid cell through the mammalian sterile 20-like kinase 1 (MST1)/Hippo pathway. METHODS: Twenty-four rats were randomly divided into 4 groups according to a random number table: control, model group, RS, and RS+Hippo inhibitor (XMU-MP-1) groups (n=6 per group). Rats were gavaged with levothyroxine sodium tablet suspension (LST, 8 μ g/kg) for 21 days except for the control group. Afterwards, rats in the RS group were gavaged with RS extracts at the dose of 1,350 mg/kg, and rats in the RS+XMU-MP-1 group were gavaged with 1,350 mg/kg RS extracts and 1 mg/kg XMU-MP-1. After 15 days of administration, thyroid gland was taken for gross observation, and histopathological changes were observed by hematoxylin-eosin staining. The structure of Golgi secretory vesicles in thyroid tissues was observed by transmission electron microscopy. The expression of thyrotropin receptor (TSH-R) was observed by immunohistochemistry. Terminal-deoxynucleoitidyl transferase mediated nick end labeling assay was used to detect cell apoptosis in thyroid tissues. Real-time quantity primer chain reaction and Western blot were used to detect the expressions of MST1, p-large tumor suppressor gene 1 (LATS1), p-Yes1 associated transcriptional regulator (YAP), proliferating cell nuclear antigen (PCNA), G₁/S-specific cyclin-D1 (Cyclin D1), B-cell lymphoma-2 (Bcl-2), Caspase-3, microtubule-associated proeins light chain 3 II/I (LC3-II/I), and recombinant human autophagy related 5 (ATG5). Thyroxine (T4) level was detected by enzyme-linked immunosorbent assay. RESULTS: The thyroid volume of rats in the model group was significantly increased compared to the normal control group (P<0.01), and pathological changes such as uneven size of follicular epithelial cells, disorderly arrangement, and irregular morphology occurred. The secretion of small vesicles by Golgi apparatus was reduced, and the expressions of receptor protein TSH-R and T4 were significantly increased (P<0.01), while the expressions of MST1, p-LATS1, p-YAP, Caspase-3, LC3-II/I, and ATG5 were significantly decreased (P<0.01). The expressions of Bcl-2, PCNA, and cyclin D1 were significantly increased (P<0.01). Compared with the model group, RS extracts reduced the volume of thyroid gland, improved pathological condition of the thyroid gland, promoted secretion of the secretory vesicles with double-layer membrane structure in thyroid Golgi, significantly inhibited the expression of TSH-R and T4 levels (P<0.01), upregulated MST1, p-LATS1, p-YAP, Caspase-3, LC3-II/I, and ATG5 expressions (P<0.01), and downregulated Bcl-2, PCNA, and Cyclin D1 expressions (P<0.01). XMU-MP-1 inhibited the intervention effects of RS extracts (P<0.01). CONCLUSION RS extracts could inhibit proliferation and promote apoptosis and autophagy in thyroid tissues through MST1/Hippo pathway for treating hyperthyroidism.
Rats ; Humans ; Animals ; Hippo Signaling Pathway ; Proliferating Cell Nuclear Antigen/metabolism* ; Cyclin D1/pharmacology* ; Caspase 3/metabolism* ; Protein Serine-Threonine Kinases/pharmacology* ; Apoptosis ; Hyperthyroidism/drug therapy* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Thyrotropin/pharmacology* ; Mammals/metabolism*

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*

Objective To investigate the neuroprotective effect of methylene blue on diabetic retinopathy in rats. Methods Thirty SD rats were randomly divided into blank, control and experimental groups. The control and experimental groups were induced with diabetes by streptozotocin (STZ) intraperitoneal injection. After 6 weeks of successful modeling, the experimental group received intravitreal injection of methylene blue at a dose of [0.2 mg/(kg.d)], while the control group received an equal amount of dimethyl sulfoxide (DMSO) intravitreal injection, both continuously injected for 7 days. ELISA was used to detect the levels of retinal superoxide dismutase (SOD), 8-iso-prostaglandin F2alpha (iPF2α) and interleukin-1β (IL-1β) in rats. Western blot analysis was used to detect the expression of retinal extracellular signal-regulated kinase 1/2 phosphorylation (p-ERK1/2) and phosphorylated protein kinase B (p-AKT), and PAS staining was used to detect retinal morphological changes. Results Compared with the blank group rats, the retinal SOD activity in the control and experimental group rats was significantly reduced. iPF2α, IL-1β and p-ERK1/2 level increased, while p-AKT level decreased. Compared with the control group, the SOD activity of the experimental group rats increased. iPF2α and IL-1β level went down, while p-ERK1/2 and p-AKT level went up significantly. The overall thickness of the retinal layer and the number of retinal ganglion cells were significantly reduced. Conclusion Methylene blue improves diabetic retinopathy in rats by reducing retinal oxidative stress and enhancing ERK1/2 and AKT phosphorylation.
Rats ; Animals ; Diabetic Retinopathy/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mitogen-Activated Protein Kinase 3/metabolism* ; Interleukin-1beta/metabolism* ; Methylene Blue/pharmacology* ; Phosphorylation ; Rats, Sprague-Dawley ; MAP Kinase Signaling System ; Diabetes Mellitus, Experimental/drug therapy* ; Superoxide Dismutase/metabolism*

This study aims to investigate the effect and mechanism of saikosaponin D on the proliferation, apoptosis, and autophagy of pancreatic cancer Panc-1 cells. The cell counting kit(CCK-8) was used to examine the effects of 7, 10, 13, 16, 19, 22, 25, and 28 μmol·L~(-1) saikosaponin D on the proliferation of Panc-1 cells. Three groups including the control(0 μmol·L~(-1)), low-concentration(10 μmol·L~(-1)) saikosaponin D, and high-concentration(16 μmol·L~(-1)) saikosaponin D groups were designed. The colony formation assay was employed to measure the effect of saikosaponin D on the colony formation rate of Panc-1 cells. The cells treated with saikosaponin D were stained with hematoxylin-eosin(HE), and the changes of cell morphology were observed. Hoechst 33258 fluorescent staining was used to detect the effect of saikosaponin D on the cell apoptosis. The autophagy staining assay kit with MDC was used to examine the effect of saikosaponin D on the autophagy of Panc-1 cells. Western blot and immunocytochemistry(ICC) were employed to examine the effect of saikosaponin D on the expression levels and distribution of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cysteine-aspartic acid protease-3(caspase-3), cleaved caspase-3, autophagy-associated protein Beclin1, microtubule-associated protein light chain 3(LC3), protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), mammalian target of rapamycin(mTOR), and phosphorylated mammalian target of rapamycin(p-mTOR). The results showed that compared with the control group, saikosaponin D decreased the proliferation rate of Panc-1 cells in a dose-dependent and time-dependent manner. The colony formation rate of the cells significantly decreased after saikosaponin D treatment. Compared with the control group, the cells treated with saikosaponin D became small, accompanied by the formation of apoptotic bodies. The saikosaponin D groups showed increased apoptosis rate and autophagic vesicle accumulation. Compared with the control group, saikosaponin D up-regulated the expression of Bax, cleaved caspase3, Beclin1, LC3Ⅱ/LC3Ⅰ and down-regulated the expression of Bcl-2, caspase-3, p-Akt/Akt, and p-mTOR/mTOR. In addition, these proteins mainly existed in the cytoplasm. In conclusion, saikosaponin D can inhibit the proliferation and induce the apoptosis and autophagy of Panc-1 cells via inhibiting the Akt/mTOR pathway.
Humans ; Proto-Oncogene Proteins c-akt/genetics* ; Caspase 3 ; bcl-2-Associated X Protein ; Beclin-1/pharmacology* ; Cell Line, Tumor ; TOR Serine-Threonine Kinases/genetics* ; Apoptosis ; Pancreatic Neoplasms/drug therapy* ; Caspases ; Autophagy

This study investigated the mechanism of Gegen Qinlian Decoction(GQD) in improving glucose metabolism in vitro and in vivo by alleviating endoplasmic reticulum stress(ERS). Molecular docking was used to predict the binding affinity between the main effective plasma components of GQD and ERS-related targets. Liver tissue samples were obtained from normal rats, high-fat-induced diabetic rats, rats treated with metformin, and rats treated with GQD. RNA and protein were extracted. qPCR was used to measure the mRNA expression of ERS marker glucose-regulated protein 78(GRP78), and unfolded protein response(UPR) genes inositol requiring enzyme 1(Ire1), activating transcription factor 6(Atf6), Atf4, C/EBP-homologous protein(Chop), and caspase-12. Western blot was used to detect the protein expression of GRP78, IRE1, protein kinase R-like ER kinase(PERK), ATF6, X-box binding protein 1(XBP1), ATF4, CHOP, caspase-12, caspase-9, and caspase-3. The calcium ion content in liver tissues was determined by the colorimetric assay. The ERS-HepG2 cell model was established in vitro by inducing with tunicamycin for 6 hours, and 2.5%, 5%, and 10% GQD-containing serum were administered for 9 hours. The glucose oxidase method was used to measure extracellular glucose levels, flow cytometry to detect cell apoptosis, glycogen staining to measure cellular glycogen content, and immunofluorescence to detect the expression of GRP78. The intracellular calcium ion content was measured by the colorimetric assay. Whereas Western blot was used to detect GRP78 and ERS-induced IRE1, PERK, ATF6, and eukaryotic translation initiation factor 2α(eIF2α) phosphorylation. Additionally, the phosphorylation levels of insulin receptor substrate 1(IRS1), phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85), and protein kinase B(Akt), which were involved in the insulin signaling pathway, were also measured. In addition, the phosphorylation levels of c-Jun N-terminal kinases(JNKs), which were involved in both the ERS and insulin signaling pathways, were measured by Western blot. Molecular docking results showed that GRP78, IRE1, PERK, ATF4, and various compounds such as baicalein, berberine, daidzein, jateorhizine, liquiritin, palmatine, puerarin and wogonoside had strong binding affinities, indicating that GQD might interfere with ERS-induced UPR. In vivo results showed that GQD down-regulated the mRNA transcription of Ire1, Atf6, Atf4, Grp78, caspase-12, and Chop in diabetic rats, and down-regulated GRP78, IRE1, PERK, as well as ERS-induced apoptotic factors ATF4 and CHOP, caspase-12, caspase-9, and caspase-3, while up-regulating XBP1 to enhance adaptive UPR. In addition, GQD increased the calcium ion content in liver tissues, which facilitated correct protein folding. In vitro results showed that GQD increased glucose consumption in ERS-induced HepG2 cells without significantly affecting cell viability, increased liver glycogen synthesis, down-regulated ATF6 and p-eIF2α(Ser51), and down-regulated IRE1, PERK, and GRP78, as well as p-IRS1(Ser312) and p-JNKs(Thr183/Tyr185), while up-regulating p-PI3Kp85(Tyr607) and p-Akt(Ser473). These findings suggested that GQD alleviates excessive ERS in the liver, reduces insulin resistance, and improves hepatic glucose metabolism in vivo and in vitro.
Rats ; Animals ; Proto-Oncogene Proteins c-akt ; Endoplasmic Reticulum Chaperone BiP ; Caspase 3 ; Caspase 9 ; Diabetes Mellitus, Experimental ; Caspase 12 ; Calcium/pharmacology* ; Molecular Docking Simulation ; Endoplasmic Reticulum Stress ; Protein Serine-Threonine Kinases/genetics* ; Liver ; Apoptosis ; Insulin ; Glucose ; Glycogen/pharmacology* ; RNA, Messenger

This study aims to investigate the therapeutic effects and potential mechanisms of resveratrol(Res) on poor ovarian response(POR) in mice. The common target genes shared by Res and POR were predicted by network pharmacology, used for Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and then validated by animal experiments. The mice with regular estrous cycle after screening were randomized into normal, POR, and low-and high-dose(20 and 40 mg·kg~(-1), respectively) Res groups. The normal group was administrated with an equal volume of 0.9% sodium chloride solution by gavage, and the mice in other groups with tripterygium glycosides suspension(50 mg·kg~(-1)) by gavage for 2 weeks. After the modeling, the mice in low-and high-dose Res groups were treated with Res by gavage for 2 weeks, and the mice in normal and POR groups with an equal volume of 0.9% sodium chloride solution by gavage. Ovulation induction and sample collection were carried out on the day following the end of treatment. Vaginal smears were collected for observation of the changes in the estrous cycle, the counting of retrieved oocytes, and the measurement of ovarian wet weight and ovarian index. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of anti-mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in the serum. The ovarian tissue morphology and granulosa cell apoptosis were observed by hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL), respectively. Western blot was employed to determine the protein levels of phosphatidylinositol 3-kinase(PI3K), protein kinase B(AKT), forkhead box O(FOXO) 3a, hypoxia-inducible factor(HIF)-1α, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). A total of 222 common targets shared by Res and POR were collected. GO annotation indicated that these targets were mainly involved in oxidative stress response. KEGG enrichment analysis revealed that Res can intervene in POR via PI3K/AKT, HIF-1, and FOXO signaling pathways. Animal experiments showed that the model group had higher rate of estrous cycle disorders, lower number and poorer morphology of normally developed follicles at all levels, more atretic follicles, higher apoptosis of ovarian granulosa cells, lower number of retrieved oocytes, lower ovarian wet weight and ovarian index, higher serum levels of FSH and LH, lower levels of AMH and E_2, higher expression levels of HIF-1α, FOXO3a and Bax, and lower expression levels of PI3K, AKT, and Bcl-2 in the ovarian tissue than the normal group. Compared with the POR group, low-and high-dose Res decreased the rate of estrous cycle disorders, improved the follicle number and morphology, reduced atretic follicles, promoted the apoptosis of ovarian granulosa cells, increased retrieved oocytes, ovarian wet weight and ovarian index, and lowered serum FSH and LH levels. Moreover, Res down-regulated the expression levels of HIF-1α, FOXO3a and Bax, and up-regulated the expression levels of PI3K, AKT and Bcl-2 in the ovarian tissue. In summary, Res can inhibit apoptosis and mitigate poor ovarian response in mice by regulating the PI3K/AKT/FOXO3a and HIF-1α pathways.
Female ; Mice ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; Resveratrol/pharmacology* ; bcl-2-Associated X Protein ; Phosphatidylinositol 3-Kinases/metabolism* ; Sodium Chloride ; Follicle Stimulating Hormone ; Proto-Oncogene Proteins c-bcl-2