The roles of autophagy, ferroptosis and pyroptosis in the anti-ovarian cancer mechanism of harmine and their crosstalk.

This study aimed to investigate the role of autophagy, ferroptosis, and pyroptosis in the antitumour mechanism of harmine (Har) and its crosstalk in ovarian cancer. By transmission electron microscopy, we found that compared with those in the control group, the cytoplasm of human ovarian cancer cells (SKOV3) treated with Har showed increased numbers of autophagic vesicles, decreased intracellular mitochondrial volume, increased bilayer membrane density, and decreased cristae. Western blot, immunofluorescence, and monodasylcadaverine (MDC) staining all suggested that Har promoted autophagy in SKOV3 cells. LY294002 and siFOXO3 rescued the inhibition of the PI3K/AKT/mTOR/FOXO3 signalling pathway and the promotion of autophagy by Har. Additionally, the levels of ferroptosis- and pyroptosis-related proteins and the levels of Fe2+ , glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) suggested that Har promoted ferroptosis and pyroptosis in SKOV3 cells. Interestingly, pretreatment with chloroquine (CQ), erastin, rapamycin (Rap), or ferrostatin-1 (Fer-1) increased or reversed the ferroptosis and pyroptosis promoted by Har, respectively. In vivo, the volume of tumours in the Har group was decreased, and immunohistochemistry revealed decreased levels of Ki-67 and GPX4 and increased levels of ATG5 and NARL3. In conclusion, Har exerts its anti-ovarian cancer effect not only by promoting autophagy by regulating the PI3K/AKT/mTOR/FOXO3 signalling pathway but also by promoting ferroptosis and pyroptosis. Additionally, there is complex crosstalk between autophagy, ferroptosis, and pyroptosis in ovarian cancer.

Characteristics of mesenchymal stem cells and their exosomes derived from giant panda (Ailuropoda melanoleuca) endometrium.

Conservation of genetic resources is an important way to protect endangered species. At present, mesenchymal stem cells (MSCs) have been isolated from the bone marrow and umbilical cords of giant pandas. However, the types and quantities of preserved cell resources were rare and limited, and none of MSCs was derived from female reproductive organs. Here, we first isolated MSCs from the endometrium of giant panda. These cells showed fibroblast morphology and expressed Sox2, Klf4, Thy1, CD73, CD105, CD44, CD49f, and CD105. Endometrium mesenchymal stem cells (eMSCs) of giant panda could induce differentiation into three germ layers in vitro. RNA-seq analysis showed that 833 genes were upregulated and 716 genes were downregulated in eMSCs compared with skin fibroblast cells. The results of GO and the KEGG analysis of differentially expressed genes (DEGs) were mainly focused on transporter activity, signal transducer activity, pathways regulating pluripotency of stem cells, MAPK signaling pathway, and PI3K-Akt signaling pathway. The genes PLCG2, FRK, JAK3, LYN, PIK3CB, JAK2, CBLB, and MET were identified as hub genes by PPI network analysis. In addition, the exosomes of eMSCs were also isolated and identified. The average diameter of exosomes was 74.26 ± 13.75 nm and highly expressed TSG101 and CD9 but did not express CALNEXIN. A total of 277 miRNAs were detected in the exosomes; the highest expression of miRNA was the has-miR-21-5p. A total of 14461 target genes of the whole miRNAs were predicted and proceeded with functional analysis. In conclusion, we successfully isolated and characterized the giant panda eMSCs and their exosomes, and analyzed their functions through bioinformatics techniques. It not only enriched the conservation types of giant panda cell resources and promoted the protection of genetic diversity, but also laid a foundation for the application of eMSCs and exosomes in the disease treatment of giant pandas.

Zuogui Wan improves trabecular bone microarchitecture in ovariectomy-induced osteoporosis rats by regulating orexin-A and orexin receptor.

OBJECTIVE: To investigate the protective effects of Zuogui Wan (ZGW) on bone loss induced by ovariectomy (OVX) and its mechanism via orexin-A and orexin receptors in the osteoporosis rat model. METHODS: Fifty Sprague-Dawley female rats were randomly divided into sham-operated (sham) group and four OVX subgroups. Rats subjected to sham and OVX were treated with the vehicle (OVX, 1 mL/100 g weight, n = 10), 17ß-estradiol (E2, 50 µg*kg-1*d-1), and ZGW at the doses of 2.3 (ZGW-L) and 4.6 (ZGW-H) g/kg/day lyophilized powder daily for 3 months, respectively. The serum biochemical parameters of 17ß-estrogen (17ß-E2), tartrate-resistant acid phosphatase (TRACP-5b) and bone alkaline phosphatase (BALP) were measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin staining was used to detect the changes in the morphological structure in bones. Microcomputed tomography was used to evaluate the bone mineral density and microarchitecture of the distal femur. The gene or protein expression of orexin-A, orexin receptor 1 (OX1R), orexin receptor 2 (OX2R), osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were assayed by either quantitative polymerase chain reaction or Western blot analysis. RESULTS: Compared with the OVX group, ZGW could reduce the serum level of TRACP-5b and increased the serum levels of BALP and17ß-E2 (P < 0.01). Meanwhile, ZGW could prevent bone loss and improved bone trabecular microarchitecture by increasing the trabeculae structure thickness and trabecular number, and arranging the trabeculae structure properly. Compared with the OVX group, it was upregulated for the orexin-A and OX2R mRNA or protein expression from the hypothalamus and tibiae, and OPG in the tibiae of ZGW groups (P < 0.01, < 0.05), while downregulated for the OX1R mRNA and protein expression in the tibiae and hypothalamus and RANKL from the tibiae (P < 0.01). CONCLUSION: ZGW exhibited a protective effect for PMOP that may be mediated via orexin-A and orexin receptors regulation.

PbrmiR397a regulates lignification during stone cell development in pear fruit.

Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA element that responded to salicylic acid to induce gene expression as confirmed using a cell-based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl- and guaiacyl-lignin monomers. Consistent with reduction in lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild-type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality.

Beneficial Phytochemicals with Anti-Tumor Potential Revealed through Metabolic Profiling of New Red Pigmented Lettuces (Lactuca sativa L.).

The present study aimed to compare polyphenols among red lettuce cultivars and identify suitable cultivars for the development and utilization of healthy vegetables. Polyphenols, mineral elements, and antioxidant activity were analyzed in the leaves of six red pigmented lettuce (Lactuca sativa L.) cultivars; thereafter, we assessed the anti-tumor effects of cultivar B-2, which displayed the highest antioxidant activity. Quadrupole-Orbitrap mass spectrometry analysis revealed four classes of polyphenols in these cultivars. The composition and contents of these metabolites varied significantly among cultivars and primarily depended on leaf color. The B-2 cultivar had the highest antioxidant potential than others because it contained the highest levels of polyphenols, especially anthocyanin, flavone, and phenolic acid; furthermore, this cultivar displayed anti-tumor effects against the human lung adenocarcinoma cell line A549, human hepatoma cell line Bel7402, human cancer colorectal adenoma cell line HCT-8, and HT-29 human colon cancer cell line. Hence, the new red-leaf lettuce cultivar B-2 has a distinct metabolite profile, with high potential for development and utilization of natural phytochemical and mineral resources in lettuces and can be used as a nutrient-dense food product.

Effects of confinement duration and parity on stereotypic behavioral and physiological responses of pregnant sows.

The aim of this study was to evaluate the effects of space restriction stress on the stereotypic behavioral and physiological responses of different parity sows, and it is necessary to understand such effects due to space restriction to improve the welfare of the sows in confined conditions. We selected fifty pregnant sows (Large White) at primiparity and first to fifth parity in a confined farm with the same body condition and due date (3±1.5days). Behavioral observations and physiological analysis were carried out during spatial confinement throughout pregnancy. The results showed that there were no significant changes in vacuum-chewing, bar-biting, trough-biting and the concentrations of serum IL-6 in primiparous sows during the initial confinement (0-8days). With the increase of the confinement duration, the serum cortisol, IgA, IL-6 concentrations and the vacuum-chewing frequency of sows in all groups increased significantly, and the serum concentrations of C-reactive protein and Pig-MAP increased significantly except for the sows in the first and second parity groups. The serum cortisol, IgA, IL-6 concentrations and the vacuum-chewing frequency of older sows were significantly higher than those of the young sows throughout the entire restricted feeding period, but the serum C-reactive protein concentrations of primiparous gilts was significantly higher than those of the other groups. The serum cortisol, IgA, IL-6 concentrations and bar-biting and trough-biting frequencies of all parity sows decreased significantly after entering the delivery bed. In conclusion, long-lasting and recurrent environmental constraints can significantly increase the frequency of stereotypical behavior and serious psychological and physical stress, seriously damaging sow welfare.

Distinct transcriptome profiles reveal gene expression patterns during fruit development and maturation in five main cultivated species of pear (Pyrus L.).

The transcriptomes of five pear cultivars, 'Hosui' (P. pyrifolia), 'Yali' (P. bretschneideri), 'Kuerlexiangli' (P. sinkiangensis), 'Nanguoli' (P. ussuriensis), and 'Starkrimson' (P. communis) were sequenced at seven key fruit developmental stages, from fruit setting to maturation and fruit senescence after harvesting. In total, 33,136 genes that could be mapped by reads, were analyzed. Most gene expression cluster models showed a steadily decreasing trend. Gene expression patterns had obvious differences according to maturity type, that is, post-ripening cultivars were still vigorous at maturity, and showed a higher proportion of up-regulated genes; non post-ripening cultivars had a gradually decreasing tendency during fruit maturation. Meanwhile, differentially expressed genes related to fruit quality and development, such as stone cells, sugar, acid and hormones, were identified. Co-expression analysis revealed that several ethylene synthesis genes and polyphenoloxidase-related genes interacted with each other directly, and an indirect relationship was reflected between ethylene synthesis genes and ethylene response genes. In addition, the highly diverse SNPs represented the great differences between oriental and occidental pears. Understanding how RNA-seq based gene-expression patterns and differential gene expression contribute to fruit quality allows us to build models for gene-expression for fruit development of Pyrus species.

Computed tomography for the diagnosis of solitary thin-walled cavity lung cancer.

BACKGROUND AND AIM: Lung cancer is the most commonly diagnosed neoplasm and the leading cause of cancer-related death worldwide. Despite the high incidence of lung cancer, the diagnosis of solitary thin-walled cavity lung cancer is rare. The aim of this review is to explore the potentials of computed tomography (CT) as diagnostic tool for solitary thin-walled cavity lung cancer. METHOD: The literature search was made in electronic databases including PudMed, Ovid SP, Embase, Web of Sciences, EBSCO and Wiley online by using relevant key terms. Because of the rarity of the subject, no precise exclusion or inclusion criteria were used for article selection and the outcome dissemination was decided to be more descriptive rather than quantitative. RESULTS: The detection of cavitation in lungs is frequently done utilizing chest radiographs CT scans. However, the diagnostic challenge remains the accurate detection of solitary thin-walled cavity lung cancer among the prevalence of cavitary lung lesions in multiple thoracic disorders including benign disorders, infectious disease and malignant tumors. Moreover, an accurate diagnosis of solitary thin-walled cavity lung cancer is further complicated by its subjective classification within the literature. In order to facilitate early diagnosis of this disease and circumvent the need for more invasive tests that may not be warranted, the overarching goal is to establish definitive radiological features of lung cavities that are indicative of malignancy. Herein, we describe the benefits of using CT to identify and diagnose solitary thin-walled cavity lung cancer, as well as explore the underlying mechanisms that contribute to thin-walled cavity formation in oncology patients. CONCLUSION: CT is the best modality for the noninvasive differentiation between malignant and nonmalignant cavities as it provides reliable information regarding the morphology and density of lesions. Besides, CT densitometry can efficiently detect the calcifications in lesions.

Shen-fu injection attenuates postresuscitation lung injury in a porcine model of cardiac arrest.

OBJECTIVE: To investigate the effects of Shen-Fu injection (SFI) on postresuscitation lung injury in a porcine model of cardiac arrest. METHODS: Twenty-four anaesthetised male Landrace pigs were subjected to 4 min of untreated ventricular fibrillation (VF), followed by standard cardiopulmonary resuscitation. Sixteen successfully resuscitated pigs were randomised into two groups (eight pigs per group); one group received an SFI infusion and the other group received a normal saline infusion, at an infusion rate of 0.24 mg/min from 15 min after the return of spontaneous circulation (ROSC) until 6h after ROSC. RESULTS: Oxygenation index, respiratory index, oxygen delivery, oxygen consumption, oxygen extraction, dynamic lung compliance, airway resistance, external vascular lung water index, and pulmonary vascular permeability index at 15 min, 30 min, 1h, 2h, 4h, and 6h after ROSC were all worse than baseline in the saline group, and were all better in the SFI group than in the saline group. The pulmonary protective effects of SFI were further confirmed by histopathological and ultrastructural observations of lung tissue. SFI infusion resulted in lower apoptosis index, caspase-3 protein expression, and malondialdehyde content of lung tissue after ROSC, and increased Bcl-2 protein expression and superoxide dismutase, Na+ -K+ -ATPase, and Ca2+ -ATPase activity compared with the saline group. CONCLUSION: Shen-Fu injection can attenuate postresuscitation lung injury through suppression of lung cell apoptosis and improvement of energy metabolism and antioxidant capacity.

Molecular mechanisms of therapeutic hypothermia on neurological function in a swine model of cardiopulmonary resuscitation.

OBJECTIVE: To explore the molecular mechanisms by which mild hypothermia following resuscitation improves neurological function in a porcine model of cardiac arrest. METHODS: Thirty-three inbred Chinese Wuzhishan (WZS) minipigs were used. After 8 min of untreated ventricular fibrillation (VF), the surviving animals (n=29) were randomly divided into two groups including serum group (n=16) and molecular group (n=13). Serum group animals were used to measure porcine-specific tumour necrosis factor-alpha (TNF-α), interleukin (IL-6, IL-10), matrix metalloproteinase (MMP9), Aquaporin-4 (AQP4), tissue inhibitor to metalloproteinase-1 (TIMP1), neuron-specific enolase (NSE) and S100B at 0.5 h, 6 h, 12 h, 24 h and 72h recovery by enzyme-linked immunosorbent assay (ELISA). Molecular group animals were used to measure cerebral cortex messenger RNA (mRNA) and protein expression of nuclear factor-κB (NF-κB), MMP9 and AQP4 by real-time (RT) quantitative polymerase chain reaction (PCR) and Western blotting at 24 h and 72 h recovery. Animals were further divided into either normothermia or hypothermia groups. Hypothermia (33°C) was maintained for 12 h using an endovascular cooling device. Swine neurologic deficit scores (NDS) were used to evaluate neurological function at 24-h and 72-h recovery. RESULTS: Twenty-nine of the 33 (87.9%) animals were successfully resuscitated. The hypothermia group exhibited higher survival rates at 24 h (75%) and 72 h (62.5%) compared to the normothermia group (37.5% and 25%, respectively). Hypothermia markedly inhibited expression of NF-κB, TNF-α, MMP9 and NSE, and promoted expression of TIMP1 (P<0.01). The mean NDS at 24-h and 72-h recovery was 112.5 and 61, respectively, in the hypothermic group, and 230 and 207.5, respectively, in the normothermia group. CONCLUSION: Brain protection induced by hypothermia involves inhibition of inflammatory and brain edema pathways.

Relation between prognosis and expression of metastasis-associated protein 1 in stage I non-small cell lung cancer.

Metastasis-associated protein 1 (MTA1 protein) has been reported to be correlated with the biological behavior and prognosis of several malignant carcinomas. We hypothesized that stage I non-small cell lung cancer (NSCLC) patients with MTA1 protein overexpression would be more likely to have a poor prognosis. Therefore, we tested the expression of MTA1 protein in 60 stage I NSCLC and 30 paracarcinous normal lung tissues using the streptavidin-perosidase method. The Kaplan-Meier method was used to calculate the survival rate, and Cox regression analysis was performed to identify prognostic risk factors. MTA1 protein overexpression was detected in 22 stage I NSCLC tissues in this study. Tumor differentiation and tumor diameter were significantly associated with MTA1 protein overexpression, while not correlated with age, sex, pathological type or smoking status. The five-year survival rate of patients with MTA1 protein overexpression was significantly lower than that of those without expression (40.9% vs. 84.1%; P<0.001). The results of multivariate analysis confirmed that MTA1 protein overexpression was an independent prognostic factor (risk ratio=5.23, P=0.007). These findings demonstrated MTA1 might be a prognostic factor in NSCLC.

Clinical study of modified Ivor-Lewis esophagectomy plus adjuvant radiotherapy for local control of stage IIA squamous cell carcinoma in the mid-thoracic esophagus.

OBJECTIVE: To control the postoperative local recurrence is one of the critical factors to improve prognosis of patients with esophageal carcinoma. The aim of this study is to evaluate the effectiveness of modified Ivor-Lewis esophagectomy plus adjuvant radiotherapy for local control of stage IIA squamous cell carcinoma in the mid-thoracic esophagus. METHODS: One hundred and twenty-five patients with stage IIA mid-thoracic esophageal squamous cell carcinoma who underwent modified Ivor-Lewis esophagectomy between June 1999 and June 2002 were included in the retrospective analysis. All the patients were evaluated within 3 years after surgery to detect tumor recurrence. Kaplan-Meier method was used to calculate the survival rate and logistic regression analysis was performed to identify risk factors of locoregional recurrence. RESULTS: The overall 3-year and 5-year survival rate in all patients was 58.4% and 43.2%, respectively. Tumor recurrence occurred in 61 patients (48.8%) within 3 years after operation. The median disease-free interval was 12.6 months. Thirty-three patients (26.4%) developed locoregional recurrence, 23 patients (18.4%) developed distant recurrence and 5 patients (4.0%) developed locoregional and distant recurrence simultaneously. Locoregional recurrence rate of patients with postoperative radiotherapy was significantly lower than that of those without postoperative radiotherapy (p<0.05). Logistic regression analysis showed that adjuvant radiotherapy (p=0.007) was an independent risk factor for tumor locoregional recurrence. Cox regression analysis showed that locoregional recurrence but not adjuvant radiotherapy was a relevant prognostic factor of patients with stage IIA esophageal cancer. CONCLUSIONS: Modified Ivor-Lewis esophagectomy with two-field lymph node dissection plus adjuvant radiotherapy might be an effective strategy to achieve local control of stage IIA mid-thoracic esophageal squamous cell carcinoma.

Abdominal lymph node metastasis in patients with mid thoracic esophageal squamous cell carcinoma.

BACKGROUND: There are few reports about abdominal lymph node metastasis of mid thoracic esophageal carcinoma. This study was designed to explore the pattern of abdominal lymph node metastasis in patients with mid thoracic esophageal squamous cell carcinoma and to evaluate the prognostic factors. METHODS: The complete data of 368 patients with mid thoracic esophageal squamous cell carcinoma, who underwent modified Ivor-Lewis esophagectomy with two-field lymphadenectomy from January 1998 to January 2003, were reviewed. Survival rate was calculated by Kaplan-Meier method. Cox regression analysis was performed to identify risk prognostic factors. RESULTS: Abdominal lymph node metastasis occurred in 58 (15.8%) patients: 34.5% (20/58) of them were stage T1 and T2. Skipping abdominal node metastasis was recognized in 13.8% (8/58) patients: all were stage T1 and T2. The overall 5-year survival rate of patients with abdominal lymph node metastasis (10.3%) was lower than that of those with thoracic node metastasis (18.3%). The prognosis of patients with distant abdominal lymph node metastasis was poor, and no one could survive more than 5 years. Cox regression analysis showed that five or more positive nodes and distant abdominal node metastasis were independent risk factors of patients with abdominal lymph node metastasis. CONCLUSIONS: Abdominal lymph node metastasis in patients with mid thoracic esophageal squamous cell carcinoma occurred frequently, and the surgery favorable for extensive abdominal lymph node dissection should be selected. The prognosis of patients with abdominal lymph node metastasis was poor, especially those with more positive nodes and distant abdominal node metastasis.

[The local control of radiotherapy following Ivor-Lewis esophagectomy in the patients with stage II A middle-third thoracic esophageal cancer].

OBJECTIVE: To investigate the local control of radiotherapy following Ivor-Lewis esophagectomy in the patients with stage IIA middle-third thoracic esophageal cancer. METHODS: From June 1999 to June 2002, 125 patients with stage IIA squamous cell carcinoma of the middle-third thoracic esophagus were treated with Ivor-Lewis esophagectomy with two-fields lymphadenectomy. The survival rate was calculated by Kaplan-meier method and the difference of recurrence rate compared by chi(2) test. RESULTS: The 3-year and 5-year survival rates were 58.4% and 43.2% in this group, respectively. Tumor recurrence occurred in 61 of the 125 patients (48.8%) within 3 years after operation. Of all cases of recurrence, 38 patients (30.4%) developed locoregional recurrence (including 5 patients with locoregional and hematogenous recurrence simultaneously). The locoregional recurrence rate of patients who received postoperative radiotherapy (20.3%) was significantly lower than that of both the group who received adjunctive chemotherapy (40.6%) and the group without adjunctive therapy (41.4%) (P < 0.05). CONCLUSIONS: About half of the patients would develop recurrence disease within 3 years after Ivor-Lewis esophagectomy with two-fields lymph-adenectomy. Radiotherapy following Ivor-Lewis esophagectomy is an effective strategy to control local recurrence of the stage II A middle-third thoracic esophageal cancer.