Analysis of abnormal expression of signaling pathways in PQ-induced acute lung injury in SD rats based on RNA-seq technology.

Background: Paraquat (PQ) plays an important role in agricultural production due to its highly effective herbicidal effect. However, it has led to multiple organ failure in those who have been poisoned, with damage most notable in the lungs and ultimately leading to death. Because of little research has been performed at the genetic level, and therefore, the specific genetic changes caused by PQ exposure are unclear.Methods: Paraquat poisoning model was constructed in Sprague Dawley (SD) rats, and SD rats were randomly divided into Control group, paraquat (PQ) poisoning group and Anthrahydroquinone-2,6-disulfonate (AH2QDS) treatment group. Then, the data was screened and quality controlled, compared with reference genes, optimized gene structure, enriched at the gene expression level, and finally, signal pathways with significantly different gene enrichment were screened.Results: This review reports on lung tissues from paraquat-intoxicated Sprague Dawley (SD) rats that were subjected to RNA-seq, the differentially expressed genes were mainly enriched in PI3K-AKT, cGMP-PKG, MAPK, Focal adhesion and other signaling pathways.Conclusion: The signaling pathways enriched with these differentially expressed genes are summarized, and the important mechanisms mediated through these pathways in acute lung injury during paraquat poisoning are outlined to identify important targets for AH2QDS treatment of acute lung injury due to paraquat exposure, information that will be used to support a subsequent in-depth study on the mechanism of PQ action.

[Effects of heavy-load exercise on skeletal muscle cells apoptosis and mechanisms of mitochondrial apoptosis in rats].

OBJECTIVE: To analyze the molecular mechanisms of skeletal muscle cells apoptosis induced by heavy-load exercise with Omi as the entry point. METHODS: One hundred and twenty-six adult SD rats were randomly divided into five groups: control group(C), eccentric exercise group (E), simple blocking group (U), DMSO group (D) and exercise block group (EU). In addition to the C group, the other four groups were randomly divided into 0 h after experiment, 12 h after experiment, 24 h after experiment, 48 h after experiment and 72 h after experiment with 6 rats in each group. E and EU group were submitted to a heavy-load exercise on a treadmill down a 16° decline, 16 m/min for 90 minutes. U, D and EU group were one-time intervened with drugs. U and EU groups were intraperitoneally injected with 1.5 µmol/kg ucf-101, D group were intraperitoneally injected with 1.5 µmoL/kg 0.5% DMSO. The rats were sacrificed in batches at different time points after experiment, then the soleus were saved to detect the Caspase-3,-8,-9,-12 activities and protein expressions of Omi and XIAP. RESULTS: Compared with group C, the mitochondrial distribution and morphology appeared the typical ultrastructure pathological changes, the opening degree of MPTP was increased significantly (P＜0.01) or (P＜0.05), protein expressions of Omi and XIAP were increased significantly (P＜0.01 or P＜0.05), the activities of Caspase-9 and Caspase-3 were increased significantly (P＜0.01 or P＜0.05) in group E. Compared with group C, there was no significant difference in XIAP protein and caspase-9, - 3 activities in group U and Group D. The change trend of XIAP protein and Caspase-9, - 3 activities was the same as those between EU group and E group, but the change range of XIAP protein in EU group was significantly higher than that in E group (P＜0.01), and the change ranges of caspase-9, - 3 activities in EU group were significantly lower than those in E group (P＜0.01). CONCLUSION: A single heavy-load exercise can induce changes in the mitochondria morphology and structure in rats, open the high permeability of MPTP, and improve the expression of Omi protein, then through its downstream XIAP-Caspase pathway, start the mitochondrial apoptosis pathway mediated by caspase-9, and finally lead to myocyte apoptosis. The inhibition of Omi can reduce the cell apoptosis level of motor induced skeletal muscle cells.

Anthrahydroquinone-2-6-disulfonate is a novel, powerful antidote for paraquat poisoning.

Paraquat (PQ) is a widely used fast-acting pyridine herbicide. Accidental ingestion or self-administration via various routes can cause severe organ damage. Currently, no effective antidote is available commercially, and the mortality rate of poisoned patients is exceptionally high. Here, the efficacy of anthrahydroquinone-2-6-disulfonate (AH2QDS) was observed in treating PQ poisoning by constructing in vivo and ex vivo models. We then explored the detoxification mechanism of AH2QDS. We demonstrated that, in a rat model, the PQ concentration in the PQ + AH2QDS group significantly decreased compared to the PQ only group. Additionally, AH2QDS protected the mitochondria of rats and A549 cells and decreased oxidative stress damage, thus improving animal survival and cell viability. Finally, the differentially expressed genes were analysed in the PQ + AH2QDS group and the PQ group by NextGen sequencing, and we verified that Nrf2's expression in the PQ + AH2QDS group was significantly higher than that in the PQ group. Our work identified that AH2QDS can detoxify PQ by reducing PQ uptake and protecting mitochondria while enhancing the body's antioxidant activity.

Peripheral cytotoxic T lymphocyte predicts first-line progression free survival in HER2-positive advanced breast cancer.

BACKGROUND: The role of peripheral blood lymphocyte (pBL) in breast cancer has long been studied. However, the predictive role of pBL in advanced breast cancer (ABC) is poorly understood. METHODS: A total of 303 patients with ABC were consecutively recruited at our center between January 2015 and September 2019. At baseline, pBL subtypes were detected in all patients with 229 blood samples available for circulating tumor DNA (ctDNA) detection. pBL was analyzed through flow cytometry. ctDNA-based gene mutations were detected using next generation sequencing. The cutoff value of pCTL was estimated by X-tile software. Progression free survival (PFS) was estimated by Kaplan-Meier curve and Cox hazard proportion regression model, with difference detection by log-rank test. RESULTS: Median follow-up time of the study was 21.0 months. The median age of diagnosis was 52.0 years. Among the pBL subtypes, only pCTL level was found predictive for PFS in the HER2+ patients whom received anti-HER2 therapy (13.1 vs. 5.6 months, P = 0.001). However, the predictive role of pCTL was not found in HR-positive (P = 0.716) and TNBC (P = 0.202). pCTL high associated with suppressive immune indictors including lower CD4/CD8 ratio (P = 0.004) and high level of Treg cell (P = 0.004). High occurrence of FGFR1 amplification which has been reported as immune suppressor was also found in HER2+ patients with pCTL high (22.2% vs. 4.3%, P = 0.048). CONCLUSIONS: Higher pCTLs level associated with shorter PFS and FGFR1 mutation in HER2+ ABC patients.

Antiapoptotic effects of velvet antler polypeptides on damaged neurons through the hypothalamic-pituitary-adrenal axis.

We investigated the effects of velvet antler polypeptide on cognitive impairment and the underlying mechanisms. Hydrogen peroxide-induced cell injury was used to establish an in vitro model of SH-SY5Y cells. In addition, we established an in vivo mouse model of cognitive impairment using intraperitoneal injections of scopolamine hydrobromide in strain mice. We administered three different doses of velvet antler polypeptide in this mouse model and assessed the influence of velvet antler polypeptide on the morphology of hippocampal neurons, hippocampal neuronal apoptosis, adrenocorticotropic hormone, and corticosterone activities in brain tissue samples, and the molecular and biochemical regulation of B-cell lymphoma-2, B-cell lymphoma-2 Associated X-protein, Cysteine-aspartic acid protease-3, glucocorticoid receptor, mineralocorticoid receptor, and corticotropin-releasing hormone in murine hippocampal neurons. Our data suggest that velvet antler polypeptide decreases glucocorticoid receptor, mineralocorticoid receptor, and corticotropin-releasing hormone levels and regulates the hormones released by the hypothalamic-pituitary-adrenal axis, thus suppressing neuronal apoptosis.

[Effects of exercises with different durations and intensities on mitochondrial autophagy and FUNDC1 expression in rat skeletal muscles].

The aim of the present study was to investigate the effects of exercises with different durations and intensities on mitochondrial autophagy and FUNDC1 in rat skeletal muscles. Sixty male Sprague-Dawley rats were randomly divided into 2- and 4-week control groups (Con), moderate-intensity exercise groups (M-ex groups, treadmill exercise, 16 m/min, 1 h/d, 6 d/week), and high-intensity exercise groups (Hi-ex groups, treadmill exercise, 35 m/min, 20 min/d, 6 d/week). The bilateral soleus muscles were separated after the intervention, and paraffin sections were prepared for transmission electron microscopy. ELISA method was used to detect the content of citrate synthase (CS). The co-localizations of microtubule-associated protein 1 light chain 3 (LC3)/cytochrome c oxidase IV (COX-IV), FUNDC1/COX-IV and LC3/FUNDC1 were observed by immunofluorescent staining in frozen sections. The skeletal muscle mitochondria were extracted, and the expression of autophagy-related proteins, including AMPKα, p-AMPKα, Unc-51 like kinase 1 (ULK1), FUNDC1, LC3 and p62, were detected by Western blot. The results showed that exercise increased mitochondrial function, i.e. peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α), COX-I protein expression levels and CS content. There was no difference of mitochondrial function parameters between 2-week M-ex and 2-week Hi-ex groups, while mitochondrial function of 4-weeks Hi-ex group was significantly lower than that of 4-week M-ex group. Under the same exercise intensity, mitochondrial autophagy activation in skeletal muscle of 4-week exercise was higher than that in 2-week exercise group; Under the same duration of exercise, mitochondrial autophagy activation of Hi-ex group was higher than that in M-ex group. Both 2- and 4-week exercise intervention increased LC3/COX-IV, COX-IV/FUNDC1, and FUNDC1/LC3 co-localizations. Exercise increased LC3-II/LC3-I ratio, down-regulated p62 protein expression level, up-regulated FUNDC1, ULK1 protein expression levels and AMPKα phosphorylation, and the changes of these proteins in 4-week Hi-ex group were significantly greater than those in 4-week M-ex group. These results suggest exercise induces mitochondrial autophagy in skeletal muscles, and the activity of autophagy is related to the duration and intensity of exercise. The induction mechanism of exercise may involve the mediation of FUNDC1 expression through AMPK-ULK1 pathway.

Methylome Variation Predicts Exemestane Resistance in Advanced ER+ Breast Cancer.

BACKGROUND: More than 30% of estrogen receptor-positive breast cancers are resistant to primary hormone therapy, and about 40% that initially respond to hormone therapy eventually acquire resistance. Although the mechanisms of hormone therapy resistance remain unclear, aberrant DNA methylation has been implicated in oncogenesis and drug resistance. PURPOSE: We investigated the relationship between methylome variations in circulating tumor DNA and exemestane resistance, to track hormone therapy efficacy. METHODS: We prospectively recruited 16 patients who were receiving first-line therapy in our center. All patients received exemestane-based hormone therapy after enrollment. We collected blood samples at baseline, first follow-up (after 2 therapeutic cycles) and at detection of disease progression. Disease that progressed within 6 months under exemestane treatment was considered exemestane resistance but was considered relatively exemestane-sensitive otherwise. We obtained circulating tumor DNA-derived methylomes using the whole-genome bisulfide sequencing method. Methylation calling was done by BISMARK software; differentially methylated regions for exemestane resistance were calculated afterward. RESULTS: Median follow-up for the 16 patients was 19.0 months. We found 7 exemestane resistance-related differentially methylated regions, located in different chromosomes, with both significantly different methylation density and methylation ratio. Baseline methylation density and methylation ratio of chromosome 6 [32400000-32599999] were both high in exemestane resistance. High baseline methylation ratios of chromosome 3 [67800000-67999999] (P = .013), chromosome 3 [140200000-140399999] (P = .037), and chromosome 12 [101200000-101399999] (P = .026) could also predict exemestane resistance. During exemestane treatment, synchronized changes in methylation density and methylation ratio in chromosome 6 [32400000-32599999] could accurately stratify patients in terms of progression-free survival (P = .000033). Cutoff values of methylation density and methylation ratio for chromosome 6 [149600000-149799999] were 0.066 and 0.076, respectively. CONCLUSION: Methylation change in chromosome 6 [149600000-149799999] is an ideal predictor of exemestane resistance with great clinical potential.

Identification of high independent prognostic value of nanotechnology based circulating tumor cell enumeration in first-line chemotherapy for metastatic breast cancer patients.

Enumeration of circulating tumor cells (CTCs) is a promising tool in the management of metastatic breast cancer (MBC). This study investigated the capturing efficiency and prognostic value of our previously reported peptide-based nanomagnetic CTC isolation system (Pep@MNPs). We counted CTCs in blood samples taken at baseline (n = 102) and later at patients' first clinical evaluation after starting firstline chemotherapy (n = 72) in a cohort of women treated for MBC. Their median follow-up was 16.3 months (range: 9.0-31.0 months). The CTC detection rate was 69.6 % for the baseline samples. Patients with ≤2 CTC/2 ml at baseline had longer median progression-free survival (PFS) than did those with >2 CTC/2 ml (17.0 months vs. 8.0 months; P = 0.002). Patients with ≤2 CTC/2 ml both at baseline and first clinical evaluation had longest PFS (18.2 months) among all patient groups (P = 0.004). Particularly, among patients with stable disease (SD; per imaging evaluation) our assay could identify those with longer PFS (P < 0.001). Patients with >2 CTC/2 ml at baseline were also significantly more likely to suffer liver metastasis (P = 0.010). This study confirmed the prognostic value of Pep@MNPs assays for MBC patients who undergo firstline chemotherapy, and offered extra stratification regarding PFS for patients with SD, and a possible indicator for patients at risk for liver metastasis.

Mammalian target of rapamycin/eukaryotic initiation factor 4F pathway regulates follicle growth and development of theca cells in mice.

The aim of the present study was to clarify the roles of the mammalian target of rapamycin (mTOR) signalling pathway in follicular growth and development of thecal cells. Using in vivo-grown and in vitro-cultured ovaries, histological changes were evaluated using haematoxylin and eosin (HE) staining. Differentially expressed genes (DEGs) from 0 day post partum (d.p.p.) to 8 d.p.p. ovaries were screened by microarray and verified by quantitative real-time polymerase chain reaction. Forty-two DEGs related to cell proliferation and differentiation were screened out, with most DEGs being related to the to mTOR signalling pathway. Then, 3 d.p.p. ovaries were retrieved and used to verify the role of mTOR signalling in follicle and thecal cell development using its activators (Ras homologue enriched in brain (Rheb) and GTP) and inhibitor (rapamycin). The development of follicles and thecal cells was significantly impaired in ovaries cultured in vitro Day 3 to Day 8. In in vitro-cultured ovaries, Rheb and GTP (is 100ngmL-1 Rheb and 500ngmL-1 GTP for 48h) significantly increased follicle diameter, the percentage of primary and secondary follicles and the umber of thecal cells, and upregulated expression of mTOR, phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), eukaryotic initiation factor (eIF) 4F and cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1). Rapamycin (10nM rapamycin for 24h) had opposite effects to those of Rheb and GTP, and partly abrogated (significant) the effects of Rheb and GTP when added to the culture in combination with these drugs. Thus, mTOR signalling plays an important role in follicle growth and thecal cell development.

Preventive effects of ß-cryptoxanthin against cadmium-induced oxidative stress in the rat testis.

ß-cryptoxanthin (CRY), a major carotenoid of potential interest for health, is obtained naturally from orange vegetables and fruits. A few research studies have reported that CRY could decrease oxidative stress and germ cell apoptosis. The purpose of this study was to examine the effects of CRY on acute cadmium chloride (CdCl 2 )-induced oxidative damage in rat testes. For this study, 24 rats were divided into four groups, one of which serves as a control group that received intraperitoneal (i.p.) injections of corn oil and physiological saline. The other rats were i.p. injected with CRY (10 µg kg-1 ) every 8 h, beginning 8 h before CdCl 2 (2.0 mg kg-1 ) treatment. The pathological and TUNEL findings revealed that CRY ameliorated the Cd-induced testicular histological changes and germ cell apoptosis in the rats. Furthermore, the Cd-induced decrease in the testicular testosterone (T) level was attenuated after CRY administration (P < 0.05). The administration of CRY significantly reversed the Cd-induced increases in the lipid peroxide (LPO) and malondialdehyde (MDA) levels (P < 0.01). The testicular antioxidants superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were decreased by treatment with Cd alone but were restored by CRY co-treatment. These results demonstrated that the application of CRY can enhance the tolerance of rats to Cd-induced oxidative damage and suggest that it has promised as a pharmacological agent to protect against Cd-induced testicular toxicity.

Anti-inflammatory potential of ß-cryptoxanthin against LPS-induced inflammation in mouse Sertoli cells.

ß-cryptoxanthin (CX), a major carotenoid pigment, can inhibit inflammatory gene expression in mice with nonalcoholic steatohepatitis. In the present study, we examined the anti-inflammatory effects of CX on lipopolysaccharide (LPS)-induced inflammation in mouse primary Sertoli cells and the possible molecular mechanisms behind its effects. The results showed that CX significantly inhibited LPS-induced decreases in cell viability and in the percentage of apoptotic cells. Moreover, CX inhibited the LPS-induced up-regulation of tumor necrosis factor α (TNF-α), interleukin-10 (IL-10), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) in Sertoli cells. In addition, CX significantly limited the LPS-induced down-regulation of AR, HSF2, CREB, FSHR, INHBB and ABP in Sertoli cells. Western blot analysis showed that CX significantly suppressed NF-κB (p65) activation as well as MAPK phosphorylation. All the results suggested that CX suppressed inflammation, possibly associated with the NF-κB activation and MAPK of phosphorylation. Thus, CX may possess therapeutic potential against inflammation-related diseases.

The Anti-Apoptotic Role of Berberine in Preimplantation Embryo In Vitro Development through Regulation of miRNA-21.

Traditional Chinese medicinal herbs containing berberine have been historically used to prevent miscarriage. Here, we investigated whether the anti-apoptotic effects of berberine on pre-implantation embryonic development are regulated by miRNA-21. Mouse pronuclear embryos were cultured in medium with or without berberine, and some were then microinjected with a miRNA-21 inhibitor. The in vitro developmental rates of 2- and 4-cell embryos and blastocysts, blastocyst cell numbers, apoptotic rates, and apoptotic cell numbers were measured in each group. Furthermore, we examined the transcription levels of miRNA-21 and its target genes (caspase-3, PTEN, and Bcl-2) and their translation levels. Comparisons were made with in vivo-developed and untreated embryos. We found that berberine significantly increased the developmental rates and cell numbers of mouse blastocysts and decreased apoptotic cell rates in vitro. Berberine also significantly increased miRNA-21 and Bcl-2 transcription levels and significantly decreased caspase-3 and PTEN transcription levels. In embryos treated with a miRNA-21 inhibitor, the results followed the opposite trend; PTEN and caspase-3 transcription levels increased significantly, while the transcription level of Bcl-2 decreased significantly. Additionally, berberine treatment significantly increased the Bcl-2 protein level and significantly decreased the caspase-3 and PTEN protein levels in blastocysts, but there were no significant differences observed in the levels of these proteins in 2- and 4-cell embryos. This study revealed that miRNA-21 is important for pre-implantation embryonic development, especially blastocyst development in vitro. Berberine elevates miRNA-21 expression, decreases PTEN and caspase-3 levels, increases Bcl-2 levels, and exerts anti-apoptotic and pro-growth effects.

The regulatory role of icariin on apoptosis in mouse preimplantation embryos with reduced microRNA-21.

We constructed a model of apoptosis in mouse preimplantation embryos and investigated the effect of the flavonol icariin on embryonic development in vitro in embryos with reduced microRNA-21 (miR-21). The model was generated by microinjecting an miR-21 inhibitor into the cytoplasm of mouse pronuclear embryos, which were cultured in vitro using modified CZB (mCZB) basal medium (model group), or using mCZB medium with 0.6 µg/mL icariin as an experimental group (model-Ica). These were compared with embryos collected in vivo (vivo group) or not microinjected (control group). Developmental rates in vitro of two- and four-cell embryos and blastocysts were observed, and Hoechst 33342 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining were used to count blastocyst cell numbers and apoptotic cell numbers and percentages. The transcriptional levels of miR-21, the apoptotic genes caspase 3 and phosphatase and tensin homolog deleted on chromosome ten (PTEN), and the antiapoptotic gene Bcl-2 were detected by quantitative polymerase chain reaction (qPCR). Western immunoblotting was used to detect the protein levels of caspase 3, PTEN, and Bcl-2. Compared with the model group, icariin treatment significantly improved blastocyst development in vitro (58.43 ± 7.53% vs. 37.85 ± 6.47%; P < 0.01), whereas it was not significantly different to the control group (60.34 ± 9.86%). Icariin treatment significantly increased the blastocyst cell numbers (47.02 ± 4.93 vs. 37.70 ± 5.80; P < 0.01), and reduced the rates of apoptosis (5.51 ± 2.35% vs. 10.11 ± 4.21%; P < 0.01), whereas the blastocyst cell numbers and apoptotic rates revealed no significant differences between the vivo (46.06 ± 6.50, 5.95 ± 2.56%) and control groups (45.77 ± 4.09, 6.18 ± 2.41%). Icariin treatment significantly improved miR-21 expression in all embryo stages, reduced the transcriptional levels of caspase 3 and PTEN, and increased the levels of Bcl-2. The protein expression levels of caspase 3 and PTEN were decreased in blastocysts and the level of Bcl-2 was increased (P < 0.01). These had no significant differences with the vivo and control groups, and the protein levels revealed no significant differences between two- and four-cell embryos. Thus, miR-21 was necessary for preimplantation embryonic development, and embryo quality was closely associated with the apoptosis-related protein expression levels regulated by miR-21. Icariin upregulated miR-21 expression and reduced apoptosis in embryos with reduced miR-21. It also improved embryonic developmental quality in vitro, indicating an important regulatory role for miR-21 in blastocyst development in vitro.