TIMP1 shapes an immunosuppressive microenvironment by regulating anoikis to promote the progression of clear cell renal cell carcinoma.

BACKGROUND: The association between ccRCC and Anoikis remains to be thoroughly investigated. METHODS: Anoikis-related clusters were identified using NMF. To identify prognostic anoikis-related genes (ARGs) and establish an optimal prognostic model, univariate Cox and LASSO regression were employed. The E-MTAB-1980 cohort was utilized for external validation. Multiple algorithms were used to evaluate the immune properties of the model. GO, KEGG and GSVA analyses were employed to analyze biological pathway functions. qRT-PCR was employed to measure RNA levels of specific genes. Cell Counting Kit-8, wound healing, and Transwell chamber assays were performed to determine changes in the proliferative and metastatic abilities of A498 and 786-O cells. RESULTS: Based on the expression of 21 prognostic ARGs, we constructed anoikis-related clusters with different prognostic and immune characteristics. The cluster A1 showed a worse prognosis, higher infiltration of immunosuppressive cells and enrichment of several oncogenic pathways. We also calculated the Anoikis Index (AI). Patients in high AI group had a worse prognosis, higher infiltration of immunosuppressive cells and higher expression of immunosuppressive checkpoints. TIMP1 exerted a tumor-promoting role in ccRCC and was significantly associated with immunosuppressive cells and checkpoints. The downregulation of TIMP1 negatively regulated ccRCC cell proliferation and metastasis. CONCLUSIONS: ARGs played crucial roles in tumorigenesis and progression and were positively associated with a poor prognosis. AI had great accuracy in predicting the prognosis and immune characteristics of ccRCC patients. TIMP1 was significantly associated with clinicopathological variables and the immunosuppressive microenvironment, which could be exploited to design novel immunotherapies for ccRCC patients.

TCN1 Deficiency Inhibits the Malignancy of Colorectal Cancer Cells by Regulating the ITGB4 Pathway.

Background/Aims: This study aimed to investigate the biological function and regulatory mechanism of TCN1 in colorectal cancer (CRC). Methods: We studied the biological function of TCN1 by performing gain-of-function and loss-of-function analyses in HCT116 cell lines; examined the effects of TCN1 on the proliferation, apoptosis, and invasion of CRC cells; and determined potential molecular mechanisms using HCT116 and SW480 CRC lines and mouse xenotransplantation models. Tumor xenograft and colonization assays were performed to detect the tumorigenicity and metastatic foci of cells in vivo. Results: TCN1 knockdown attenuated CRC cell proliferation and invasion and promoted cell apoptosis. Overexpression of TCN1 yielded the opposite effects. In addition, TCN1-knockdown HCT116 cells failed to form metastatic foci in the peritoneum after intravenous injection. Molecular mechanism analyses showed that TCN1 interacted with integrin subunit ß4 (ITGB4) to positively regulate the expression of ITGB4. TCN1 knockdown promoted the degradation of ITGB4 and increased the instability of ITGB4 and filamin A. Downregulation of ITGB4 at the protein level resulted in the disassociation of the ITGB4/plectin complex, leading to cytoskeletal damage. Conclusions: TCN1 might play an oncogenic role in CRC by regulating the ITGB4 signaling pathway.

Clinicopathological Analysis and Prognostic Assessment of TCN1 in Patients with Gastric Cancer.

BACKGROUND: Stomach cancer is the fourth most common type of cancer worldwide. TCN1 mainly encodes the vitamin B12 transporter, transcobalamin. TCN1 is a marker of gastrointestinal tumor progression, but the impact of TCN1 on survival is unclear. MATERIAL/METHODS: Gastrointestinal tumor records were reviewed and analyzed, clinicopathological data were summarized, immunohistochemical detection of TCN1 was performed again, and the protein expression in tumor tissue, non-tumor tissue, and lymph nodes was semi-quantitatively analyzed. Patients were followed up for 5 years to determine the 5-year survival rates. RESULTS: The strong immune reactivity of the TCN1 protein was significantly correlated with tumor invasion depth, regional lymph nodes, and a tumor diameter of >5 cm (Z = -2.531 and P = .016; Z = 3.785 and P < .001; Z = 2.541 and P = .049). Kaplan-Meier survival analysis showed that the total survival time of patients in the low-expression TCN1 group was significantly longer than that in the high-expression TCN1 group (P = .001; Table 2 and Figure 5). The mean survival time of all patients was 49.774 months (95% CI: 47.871-51.676; Table 4) and the 5-year overall survival rates were 73.3, 50.8, and 34.0%, respectively. Multivariate analysis revealed that regional lymph nodes (HR = 1.253; 95% CI: 1.031-1.747, P = .012), TCN1 immune expression status (HR = 2.707; 95% CI: 1.068-1.886, P = .016), and pTNM staging (HR = 2.293; 95% CI: 1.583-3.321; P = .001) were independent risk factors for poor survival. CONCLUSION: The high expression of TCN1 in gastric tumor tissues was found to be associated with the clinicopathological factors of patients, and the high expression of TCN1 was shown to indicate a poor clinical prognosis.

Uncoupling protein 1 knockout aggravates isoproterenol-induced acute myocardial ischemia via AMPK/mTOR/PPARα pathways in rats.

Uncoupling protein 1 (UCP1) was found exclusively in the inner membranes of the mitochondria of brown adipose tissue (BAT). We found that UCP1 was also expressed in heart tissue and significantly upregulated in isoproterenol (ISO)-induced acute myocardial ischemia (AMI) rat model. The present study is to determine the underlying mechanism involved in the UCP1 upregulation in ISO-induced AMI rat model. The Ucp1-/- rats were generated by CRISPR-Cas9 system and presented decreased BAT volume. 2-months old Sprague Dawley (SD) wild-type (WT) and Ucp1-/- rats were treated with ISO intraperitoneally 30 mg/kg once a day for 3 consecutive days to establish AMI model. In saline group, the echocardiographic parameters, serum markers of myocardial injury cardiac troponin I (cTnI), creatine kinase isoenzyme MB (CK-MB), oxidant malondialdehyde (MDA), antioxidant superoxide dismutase (SOD) or fibrosis were comparable between WT and Ucp1-/- rats. ISO treatment induced worse left ventricle (LV) hypertrophy, myocardial fibrosis, increased higher cTnI, CK-MB and MDA and decreased lower SOD level in Ucp1-/- rats compared with that of WT rats. Ucp1-/- rats also presented lower myocardial phosphocreatine (PCr)/ATP-ratio, which demonstrated worse cardiac energy regulation defect. ISO treatment induced the phosphorylation of AMP-activated protein kinase (AMPK) activation, subsequently the phosphorylation of mammalian target of rapamycin (mTOR) inhibition and peroxisome proliferators-activated receptor α (PPARα) activation in WT rats, whereas activation of AMPK/mTOR/PPARα pathways significantly inhibited in Ucp1-/- rats. To sum up, UCP1 knockout aggravated ISO-induced AMI by inhibiting AMPK/mTOR/PPARα pathways in rats. Increasing UCP1 expression in heart tissue may be a cytoprotective therapeutic strategy for AMI.

Effect of CRRT combined with low-flow ECMO on canines with ARDS and hypercapnia.

To observe the effect of continuous renal replacement therapy (CRRT) combined with low-flow extracorporeal membrane oxygenation (ECMO) of V-V mode on anti-inflammation, improving oxygenation and reducing PaCO2 in canines with acute respiratory distress syndrome (ARDS) and hypercapnia. A total of 30 healthy adult canines were randomly divided into sham group (n = 10), ECMO (EC) group (n = 10) and CRRT + ECMO (CR + EC) group (n = 10). Sham group was only treated with invasive mechanical ventilation. EC group was also treated with ECMO. CR + EC group was treated with CRRT combined with low-flow ECMO of V-V mode besides invasive mechanical ventilation. The results showed that hazard ratio was lower in the CR + EC group. Inflammatory factors, OI values, and PaCO2 levels were lower in the CR + EC group. There was no significant difference in the levels of MAP, CO and T among the three groups. No significant complications or death was developed in the three groups. Compared with ECMO group at T3, T6 and T9, IL-6 [(276.13 ± 8.32, 262.04 ± 7.15, 259.33 ± 7.31)ng/L VS (352.67 ± 19.24, 360.24 ± 23.58, 362.21 ± 25.24)ng/L] and TNF-α [(50.14 ± 1.75, 50.45 ± 1.81, 48.03 ± 1.24) ng/L VS (70.25 ± 3.02, 72.45 ± 3.25, 76.69 ± 2.18)ng/L] in CR + EC group were decreased (P < 0.0001). Compared with sham group, IL-6 [(343.76 ± 21.97, 345.91 ± 19.89, 340.34 ± 22.17)ng/L]and TNF-α [(68.10 ± 2.96, 67.31 ± 3.01, 70.34 ± 3.35)ng/L] of T3, T6 and T9 in CR + EC group were lower (P < 0.0001). These findings indicated that CRRT combined with low-flow ECMO of V-V mode had a positive effect on anti-inflammation, oxygenation improvement and surplus blood CO2 removal in canines with ARDS and hypercapnia. These results provide a promising treatment regimen for ARDS.

Curcumol increases the sensitivity of colon cancer to 5-FU by regulating Wnt/ß-catenin signaling.

BACKGROUND: 5-fluorouracil (5-FU) resistance is the leading cause of treatment failure in colon cancer. Combination therapy is an effective strategy to inhibit cancer cells and prevent drug resistance. Therefore, we studied the antitumor effect of curcumol alone or combined with 5-FU on human colon cancer drug-resistant cells. METHODS: The 5-FU resistant HCT116 cell line (HCT116/5-FU) was established by repeated exposure to gradually increasing concentrations of 5-FU; Cell viability was measured by cell counting kit-8 (CCK-8); apoptosis rate of HCT116 cells was detected using Annexin V-fluorescein isothiocyanate (FITC) assay kit; cell proliferation and invasion were detected using colony formation assays, wound healing assay and transwell invasion assays; activity of transplanted tumor in vivo in specific pathogen free (SPF) BALB/c nude mice (6 weeks old, male) was monitored by bioluminescence imaging, immunohistochemistry and western blot analysis. RESULTS: Our study showed the potent antitumor effect of curcumol by induction of apoptosis, inhibition of proliferation, invasion, migration, and improvement of the therapeutic efficacy of 5-FU toward human colon cancer HCT116 cells. From our results, curcumol could chemosensitize 5-FU-resistant HCT116 cells. The combination of curcumol and 5-FU exerted a synergistic inhibitory effect on the induction of apoptosis. Also, this combination inhibited the proliferation, invasion, and migration of both chemo-resistant and sensitive cells. Curcumol treatment decreased multidrug resistance-associated protein 2 (MRP-2), P-glycoprotein (P-gp), survivin, and ß-catenin expression, which correlated with multidrug resistance (MDR) and the target genes of Wnt/ß-catenin. It significantly increased the p-ß-catenin level and Bad/Bcl-2 ratio in HCT116/5-FU cells compared with 5-FU treatment. In vivo, curcumol significantly inhibited the growth of transplanted tumors and the expression of Ki-67, proliferating cell nuclear antigen (PCNA), and vascular endothelial growth factor (VEGF) in colon cancer cells. CONCLUSIONS: Curcumol as a potential chemotherapeutic agent combined with 5-FU can overcome colon cancer resistance.

CD47 antibody suppresses isoproterenol-induced cardiac hypertrophy through activation of autophagy.

In this study, we investigated whether CD47 antibody could attenuate isoproterenol (ISO)-induced cardiac hypertrophy in mice and H9C2 cells. Cardiac hypertrophy was induced by intraperitoneal (i.p.) injection of ISO (60 mg.kg-1.d-1 in 100 µl of sterile normal saline) daily for 14 days, and cell hypertrophy was induced by ISO (10-5 mol/l) for 48 h. The injury was confirmed by increased levels of lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB), increased heart-to-body weight (HW/BW) ratio and visible cardiac fibrosis. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Autophagic flux in H9c2 cells was monitored by TEM and mRFP-GFP-LC3 virus transfection. The expression levels of Cleaved caspase-3, Cleaved caspase-9 and autophagy-related proteins were detected by Western blotting. CD47 antibody significantly limited ISO-induced increases in LDH, CK-MB, HW/BW ratio and attenuated cardiac fibrosis, oxidative stress, and apoptosis in the heart; CD47 antibody promoted autophagy flow and decreased cell apoptosis in cardiac tissues. Moreover, autophagy inhibitor chloroquine (CQ) reversed the effect of CD47 antibody treatment. In conclusion, CD47 antibody reduced ISO-induced cardiac hypertrophy by improving autophagy flux and rescuing autophagic clearance.

Clinicopathological Analysis and Prognostic Assessment of Transcobalamin I (TCN1) in Patients with Colorectal Tumors.

BACKGROUND Colorectal cancer (CRC) is one of the most common malignancies worldwide. Overall survival (OS) of patients is largely dependent on disease stage at diagnosis and/or surgical resection. TCN1 mainly encodes the vitamin B12 transporter, transcobalamin. Early studies show that TCN1 is a marker of CRC progression, but the impact of TCN1 on survival is unclear. MATERIAL AND METHODS We reviewed and analyzed colorectal tumor records, summarized the clinicopathological data, performed immunohistochemical detection of TCN1 again, and semi-quantitatively analyzed protein expression in tumor tissue, non-tumor tissue, and lymph nodes. We followed up patients for 5-year survival. RESULTS Of 123 patients, 60 (48.7%) had a strong TCN1 immunohistochemical reaction, 36 (29.3%) had a moderate immune response, and 27 (22.0%) had weak expression. The level of immunohistochemical reactivity of TCN1 was correlated with the degree of histological differentiation (H (2.92)=4.976; P=0.083). Survival analysis showed that OS in patients with low TCN1 expression was significantly longer than that in the medium and high TCN1 expression groups (P=0.045). Five-year OS in patients with low, medium, and high TCN1 expression was 88.9%, 50.0%, and 40.0%, respectively. In univariate analysis, TCN1 immune expression was significantly correlated with the 5-year survival rate. CONCLUSIONS Although independent risk factors affecting survival of patients with CRC are age, serum CA125, CA19-9, lymph node metastasis, and nerve invasion, negative factors affecting overall 5-year survival in TCN1 should not be ignored, because its high expression suggests a worse clinical prognosis.

CD47 Deficiency Attenuates Isoproterenol-Induced Cardiac Remodeling in Mice.

In this study, we investigated whether CD47 deficiency attenuates isoproterenol- (ISO-) induced cardiac remodeling in mice. Cardiac remodeling was induced by intraperitoneal (i.p.) injection of ISO (60 mg·kg-1·d-1 in 100 µl of sterile normal saline) daily for 14 days and was confirmed by increased levels of lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB), increased heart weight to body weight (HW/BW) ratios, and visible cardiac fibrosis. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Levels of malondialdehyde (MDA) and reactive oxygen species (ROS) were found to be significantly higher in the ISO group than in the control group, while superoxide dismutase (SOD) levels were suppressed in the ISO group. However, CD47 knockout significantly limited ISO-induced increases in LDH, CK-MB, and HW/BW ratios, cardiac fibrosis, oxidative stress, and apoptosis in the heart. In addition, CD47 deficiency also increased p-AMPK and LAMP2 expression and decreased HDAC3, cleaved Caspase-3, cleaved Caspase-9, LC3II, and p62 expression in cardiac tissues. In conclusion, CD47 deficiency reduced i.p. ISO-induced cardiac remodeling probably by inhibiting the HDAC3 pathway, improving AMPK signaling and autophagy flux, and rescuing autophagic clearance.

Tanshinone IIA attenuates cerebral aneurysm formation by inhibiting the NF­κB­mediated inflammatory response.

The inflammatory response plays a vital role in cerebral aneurysm (CA) formation and progression. Tanshinone  IIA (Tan IIA) is one of the major active components of Chinese medicine Danshen (Salvia miltiorrhiza Bunge) and is widely used for the treatment of cardiovascular diseases, due to its anti­inflammatory effects. The aim of the present study was to investigate whether Tan IIA can attenuate CA formation in rat models, and determine its underlying mechanisms. CAs were induced in rats surgically and through high­salt diet treatments. The Tan IIA­treated group displayed relatively mild symptoms, as compared with the control group. Tan IIA treatment reduced macrophage infiltration and nuclear factor (NF)­κB activation in aneurysmal walls. Next, lipopolysaccharide (LPS)­stimulated RAW 264.7 murine macrophage cells were used to examine the anti­inflammatory effects of Tan IIA on macrophages. It was found that Tan IIA reversed LPS­induced differentiation of RAW 264.7 cells and suppressed NF­κB pathway activation. In conclusion, these findings demonstrated that Tan IIA can suppress CA formation by inhibiting inflammatory responses in macrophages.

CD47 deficiency protects cardiomyocytes against hypoxia/reoxygenation injury by rescuing autophagic clearance.

To assess the effect of cluster of differentiation (CD47) downregulation on autophagy in hypoxia/reoxygenation (H/R)­treated H9c2 cardiomyocytes. H9c2 cells were maintained in normoxic conditions (95% air, 5% CO2, 37˚C) without CD47 antibodies, Si­CD47 or chloroquine (CQ) treatment; H9c2 cells in the H/R group were subjected to 24 h of hypoxia (1% O2, 94% N2, 5% CO2, 37˚C) followed by 12 h of reoxygenation (95% air, 5% CO2, 37˚C). All assays were controlled, triplicated and repeated on three separately initiated cultures. The biochemical parameters in the medium supernatant were measured to evaluate the oxidative stress in cardiomyocytes. The Annexin V­fluorescein isothiocyanate assay was used to detect the apoptotic rate in the H9c2 cells. Transmission electron microscope, immunofluorescent staining and western blot analysis were performed to detect the effect of the CD47 antibody on autophagic flux in H/R­treated H9c2 cardiomyocytes. The cardiomyocytic oxidative stress and apoptotic rate decreased and autophagic clearance increased after CD47 downregulation. H/R triggered cell autophagy, autophagosome accumulation and apoptosis in H9c2 cell lines. However, these effects can be attenuated by CD47 downregulation. This study demonstrates its clinical implications in ischemia/reperfusion injury treatment.

Derivation of novel naive-like porcine embryonic stem cells by a reprogramming factor-assisted strategy.

The establishment of ungulate embryonic stem cells (ESCs) has been notoriously difficult via a conventional approach. We combined a traditional ESC culture method with reprogramming factors to assist the establishment of porcine naive-like ESCs (nESCs). Pig embryonic fibroblasts were transfected with a tetracycline-inducible vector carrying 4 classic mouse reprogramming factors, followed by somatic cell nuclear transfer and culturing to the blastocyst stage. Then, the inner cell mass was isolated and seeded in culture medium. The naive-like ESCs had characteristic verys similar to those of mouse ESCs and showed no signs of altered morphology or differentiation, even after 130 passages. They depended on leukemia inhibitory factor signals for maintenance of pluripotency, and the female cell lines had low expression of the X-inactive specific transcript gene and no histone H3 lysine 27 trimethylation spot. Notably, the ESCs differentiated into 3 germ layers in vitro and could be induced to undergo directional neural and kidney precursor differentiation under defined conditions, and the ESCs could keep proliferating after doxycycline was removed. nESCs can be established, and the well-characterized ESC lines will be useful for the research of transgenic pig models for human disease.-Zhang, M., Wang, C., Jiang, H., Liu, M., Yang, N., Zhao, L., Hou, D., Jin, Y., Chen, Q., Chen, Y., Wang, J., Dai, Y., Li, R. Derivation of novel naive-like porcine embryonic stem cells by a reprogramming factor-assisted strategy.

Roles of HIF1α- and HIF2α-regulated BNIP3 in hypoxia-induced injury of neurons.

BACKGROUND: To explore the roles of HIF1α- and HIF2α-regulated BNIP3 in hypoxia-induced injury of neurons. METHODS: The sera of neonates with hypoxic-ischemic encephalopathy (HIE) within 24 h after birth and full-term healthy newborns (n = 40) were collected. The BNIP3 levels were detected by ELISA. AGE1.HN cells were cultured in 1% O2 at 37 °C. The apoptosis of cells treated with 1, 5 and 10 ng/ml BNIP3 for 48 h was detected by flow cytometry. The proliferation of cells transfected with siBNIP3 was detected by CCK-8 assay. The mRNA level of BNIP3 in cells under hypoxic conditions was measured by RT-PCR. The protein level of BNIP3 in cells cultured under hypoxic conditions after pretreatment with HIF1α or HIF2α inhibitor was measured by Western blot. RESULTS: The serum BNIP3 concentration of HIE neonates ((4.5 ± 2.1) ng/ml) was significantly higher than that of healthy neonates ((1.2 ± 0.5) ng/ml) (P < 0.001). Compared with untreated group, the number of apoptotic AGE1.HN cells treated with BNIP3 significantly increased (P < 0.05). Under hypoxic conditions (1%), the mRNA and protein levels of BNIP3 increased significantly with prolonged time. After pretreatment with HIF1α or HIF2α inhibitor and hypoxic culture, BNIP3 expression was significantly lower than that of cells hypoxically cultured only. Inhibiting the expression of HIF1α or HIF2α or transfecting with siBNIP3 before hypoxic treatment significantly reduced the number of apoptotic cells. Under hypoxic conditions, HIF1α or HIF2α bound BNIP3 promoter, which did not occur under normal culture conditions. HIF1α or HIF2α was significantly enriched near the hypoxia response element (HRE) site of BNIP3 promoter. CONCLUSIONS: BNIP3 was involved in the apoptosis of cells undergoing HIE. The HRE site of BNIP3 promoter bound HIF to promote its transcription.

Effect of Hyperin and Icariin on steroid hormone secretion in rat ovarian granulosa cells.

AIM OF THE STUDY: This study was designed to investigate the effect of different concentrations of Hyperin and Icariin (ICA)on proliferation and the secretion of estrogen (E2), and progesterone (P) in granulosa cells, and to explore the effect of Hyperin and Icariin on the expression of CYP17 and CYP19. MATERIALS AND METHODS: Rat ovary granulosa cells were cultured in vitro and treated with different concentrations of Hyperin and Icariin. The proliferation of ovarian granulosa cells was measured with the MTT assay. The concentration of estradiol was measured with a magnetic particle-based enzyme-linked immunosorbent assay (ELISA) kit. The CYP17 and CYP19 mRNA expression was detected by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). The CYP17 and CYP19 protein expression was determined with Western blotting. RESULTS: Hyperin (50 µg/l) and Icariin (10 µg/l) significantly increased proliferation of ovarian granulosa cells and secretion of estrogen and progesterone. Hyperin and Icariin stimulated the mRNA and protein expression of CYP17 and CYP19. CONCLUSIONS: These results showed that Hyperin and Icariin can promote the secretion of E2 and P through up-regulation of CYP17 and CYP19. Frequently used Chinese herbs like Cuscuta Chinensis Lam and Epimedium Brevicornu maxim, which contain Hyperin and Icariin, could improve the ovarian endocrine function through these effects.

Hydralazine protects against renal ischemia-reperfusion injury in rats.

In this study, we investigated whether hydralazine could reduce renal ischemia and reperfusion (I/R) injury in rats. Renal I/R was induced by a 70-min occlusion of the bilateral renal arteries and a 24-h reperfusion, which was confirmed by the increased the mortality, the levels of blood urea nitrogen (BUN), blood creatinine (Cr), renal tissue NO and the visible histological damage of the kidneys. Apoptosis was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) staining. Furthermore, the serum levels of malonaldehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) were significantly elevated in renal I/R group, while the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) levels were suppressed. However, intragastric pretreatment with hydralazine at doses of 7.5-30 mg/kg before renal I/R significantly limited the increase in mortality, BUN, Cr, oxidative stress, inflammatory factors, histological damage and apoptosis in the kidneys. In addition, hydralazine also increased p-AKT, Bcl-2 expression and decreased iNOS, Bax, cleaved caspase-3 expression in the kidneys. In conclusion, hydralazine reduced renal I/R injury probably via inhibiting NO production by iNOS/NO pathway, inhibiting oxidative stress, inflammatory response and apoptosis by a mitochondrial-dependent pathway.

Establishment of a Mouse Model of Premature Ovarian Failure Using Consecutive Superovulation.

BACKGROUND/AIMS: This study investigated the effect of consecutive superovulation on the ovaries and established a premature ovarian failure (POF) model in mice. METHODS: The mouse POF model was induced by 5-15 consecutive superovulation treatments with pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (HCG) and prostaglandin F2α (PGF2α). Normal adult mice were compared with mice displaying natural ovarian aging. The following serum biochemical parameters were measured: including follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P), estradiol (E2), inhibin B (INH B), malondialdehyde (MDA), total superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels. Follicles were counted using H&E staining. Levels of 8-hydroxyguanosine (8-OhdG), 4-hydroxynonenal (4-HNE), nitrotyrosine (NTY), anti-Mullerian hormone (AMH) and CDKN2A/ p16 (p16) were detected using immunohistochemical staining. Reactive oxygen species (ROS) levels were measured using dihydroethidium (DHE) staining. Cell apoptosis was detected using an in situ TUNEL fluorescence staining assay. Levels of proteins involved in ROS-related pathways and the p16 protein were detected using Western blotting. Sod1, Sod2 and Sod3 mRNA levels were detected using quantitative polymerase chain reaction (Q-PCR). Oocyte quality was evaluated using in vitro fertilization (IVF) and zygote culture. RESULTS: Consecutive superovulation groups presented lower P, E2, SOD, GSH-Px and INH B levels, significantly higher FSH, LH, MDA and ROS levels, and significantly fewer primordial follicles compared with the control group. Consecutive superovulation groups presented significantly increased levels of Sod2, 8-OhdG, 4-HNE, NTY, significantly increased levels of the SIRT1 and FOXO1 proteins, significantly increased levels of the senescence-associated protein p16, as well as decreased AMH, Sod1 and Sod3 levels and increased granulosa cell apoptosis compared with the control group. CONCLUSION: Consecutive superovulation significantly decreased ovarian function and oocyte quality and increased oxidative stress and apoptosis in the ovary via a mechanism involving the p16 and SIRT1/FOXO1 signaling pathways. These findings suggest that consecutive superovulation may be used to establish a mouse model of ovarian aging.