AFB1 Triggers Lipid Metabolism Disorders through the PI3K/Akt Pathway and Mediates Apoptosis Leading to Hepatotoxicity.

As the most prevalent mycotoxin in agricultural products, aflatoxin B1 not only causes significant economic losses but also poses a substantial threat to human and animal health. AFB1 has been shown to increase the risk of hepatocellular carcinoma (HCC) but the underlying mechanism is not thoroughly researched. Here, we explored the toxicity mechanism of AFB1 on human hepatocytes following low-dose exposure based on transcriptomics and lipidomics. Apoptosis-related pathways were significantly upregulated after AFB1 exposure in all three hES-Hep, HepaRG, and HepG2 hepatogenic cell lines. By conducting a comparative analysis with the TCGA-LIHC database, four biomarkers (MTCH1, PPM1D, TP53I3, and UBC) shared by AFB1 and HCC were identified (hazard ratio > 1), which can be used to monitor the degree of AFB1-induced hepatotoxicity. Simultaneously, AFB1 induced abnormal metabolism of glycerolipids, sphingolipids, and glycerophospholipids in HepG2 cells (FDR < 0.05, impact > 0.1). Furthermore, combined analysis revealed strong regulatory effects between PIK3R1 and sphingolipids (correlation coefficient > 0.9), suggesting potential mediation by the phosphatidylinositol 3 kinase (PI3K) /protein kinase B (AKT) signaling pathway within mitochondria. This study revealed the dysregulation of lipid metabolism induced by AFB1 and found novel target genes associated with AFB-induced HCC development, providing reliable evidence for elucidating the hepatotoxicity of AFB as well as assessing food safety risks.

Synergistic effects on oxidative stress, apoptosis and necrosis resulting from combined toxicity of three commonly used pesticides on HepG2 cells.

The widespread use of pesticides performs a vital role in safeguarding crop yields and quality, providing the opportunity for multiple pesticides to co-exist, which poses a significant potential risk to human health. To assess the toxic effects caused by exposures to individual pesticides (chlorpyrifos, carbofuran and acetamiprid), binary combinations and ternary combinations, individual and combined exposure models were developed using HepG2 cells and the types of combined effects of pesticide mixtures were assessed using concentration addition (CA), independent action (IA) and combination index (CI) models, respectively, and the expression of biomarkers related to oxidative stress, apoptosis and cell necrosis was further examined. Our results showed that both individual pesticides and mixtures exerted toxic effects on HepG2 cells. The CI model indicated that the toxic effects of pesticide mixtures exhibited synergistic effects. The results of the lactate dehydrogenase (LDH) release and apoptosis assay revealed that the pesticide mixture increased the release of LDH and apoptosis levels. Moreover, our results also showed that individual pesticides and mixtures disrupted redox homeostasis and that pesticide mixtures produced more intense oxidative stress effects. In conclusion, we have illustrated the enhanced combined toxicity of pesticide mixtures by in-vitro experiments, which provides a theoretical basis and scientific basis for further toxicological studies.

Medicarpin Protects Cerebral Microvascular Endothelial Cells Against Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury via the PI3K/Akt/FoxO Pathway: A Study of Network Pharmacology Analysis and Experimental Validation.

Medicarpin, a pterocarpan class of naturally occurring phytoestrogen possesses various biological functions. However, the effect of medicarpin on oxygen-glucose deprivation-reoxygenation (OGD/R)-induced injury in human cerebral microvascular endothelial cells (HCMECs) remains largely unknown. Target genes of medicarpin were predicted from PharmMapper. Target genes of ischemic stroke were predicted from public databases GeneCards and DisGeNET. Kyoto Encyclopedia of Genes and Genomes pathway enrichment of the intersecting targets was analyzed via DAVID 6.8. Cell viability was evaluated using CCK-8 assay. Malondialdehyde content, superoxide dismutase activity, and glutathione level were detected using corresponding commercially available kits. Cell death was assessed by TUNEL assays. Expression of protein kinase B (Akt), phosphorylated-Akt, forkhead box protein O1, phosphorylated-FoxO1, FoxO3a, and phosphorylated-FoxO3a (p-FoxO3a) was detected by western blot analysis. The intersecting targets of medicarpin and ischemic stroke were significantly enriched in phosphatidylinositol 3-kinase (PI3K)/Akt and FoxO pathways. Medicarpina attenuated OGD/R-evoked viability inhibition, oxidative stress, and cell death in HCMECs. Additionally, medicarpin activated the PI3K/Akt and FoxO pathways in OGD/R-induced HCMECs. Inhibition of PI3K/Akt pathway abrogated the neuroprotective effect of medicarpin on OGD/R-induced injury and activation of FoxO pathway in HCMECs. In conclusion, medicarpin suppressed OGD/R-induced injury in HCMECs by activating PI3K/Akt/FoxO pathway.

Three widely used pesticides and their mixtures induced cytotoxicity and apoptosis through the ROS-related caspase pathway in HepG2 cells.

Difenoconazole, cypermethrin and triazophos are widely used pesticides in agricultural production and frequently detected in foods. The aim of this study was to determine the effect of these pesticides and their mixtures on cell viability, reactive oxygen species (ROS), lactate dehydrogenase (LDH) content, apoptosis rate and DNA fragmentation and synthesis in human hepatocellular carcinoma cells (HepG2). The order of inhibitory effects for the individual pesticides was ranked as difenoconazole > cypermethrin > triazophos. The enhanced expression of caspase-3, caspase-7 and PARP activity was observed in HepG2 cells, which was 1.7, 1.3 and 1.6-fold higher than the control, respectively, along with significant protein cleavage; and induced apoptosis in a concentration-dependent manner. Further, the pesticide mixtures significantly increased ROS level (up to 1.3-fold), induced DNA fragmentation (up to 1.8-fold), inhibited DNA synthesis (up to 53%), and damaged the cells by destroying the cell membrane and producing a large amount of LDH at concentration range of 10-30 µM. Specifically, mixtures containing difenoconazole showed stronger toxicities than individual pesticides, implying higher health risks associated with mixtures. Our results show that three widely used pesticides exhibited cytotoxicity and apoptosis through the ROS-related caspase pathway, providing a basis for evaluation of health risks from pesticide mixtures via food consumption.

Combined anti-androgenic effects of mixtures of agricultural pesticides using in vitro and in silico methods.

Humans and wildlife are continuously and simultaneously exposed to various pesticides that have been identified as endocrine disruptors which interfere with regulations of sexual differentiation and fertility. Low-dose effects of combined exposure from mixtures of pesticides have been extensively reported and need to be addressed in the context of human health risk assessment. The objective of the study is to assess the individual and combined anti-androgenic effects of twelve widely used pesticides in MDA-kb2 cells. The order of potency for seven pesticides with moderate anti-androgenic activities was in the order: fenitrothion > dimethomorph > difenoconazole > bromopropylate > prochloraz > imazalil > endosulfan, which was induced by the androgen receptor (AR) antagonism rather than cytotoxicity (with the exception of endosulfan which exhibited the highest cytotoxicity). The other five pesticides exhibited lower anti-androgenic activities. At 10% of AR antagonistic effect, three mixtures comprised of the seven pesticides (Mix-EC10, Mix-EC20, and Mix-EC25) at equi-effect concentrations showed summed concentrations of 6.75E-11, 17.63 and 25.21 µM, respectively. The combined effects were essentially close to the predicted of concentration addition (CA) at realistically low concentrations. In addition, molecular docking simulation indicated that hydrophobic interaction and polar functional groups of the pesticides contributed to the binding energy, which might be responsible for the AR antagonism. Our findings provide a basis for defining similarly acting antagonists in the context of cumulative risk assessment for pesticides in foods.

PD98059 inhibited the activation of pancreatic stellate cells mediated by platelet-derived growth factor BB in rats.

To determine the biological effects of extracelluar signal regulated kinase (ERK) specific inhibitor PD98059 on pancreatic stellate cells (PSCs) activated by platelet-derived factor-BB (PDGF-BB), cultured rat PSCs were co-incubated at 37 DC for 24 h with 25 ng/ml PDGF-BB and different doses of PD98059 (ranging from 5 ng/ml to 40 ng/ml). Expression of pERK1 protein was detected by Western blot and collagen alpha1 ( I ) mRNA was tested by reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that there were statistical differences in the expression of pERK1 protein in all groups. Expression of pERK1 protein was up-regulated in the group treated by PDGF-BB, and gradually down-regulated in the other groups treated by PD98059 of different doses. An excellent positive correlation was revealed between the inhibitory effect and PD98059 concentrations. It was also observed that the expression of collagen alpha1 ( I ) mRNA had similar response to pERK1. The level of collagen alpha1 ( I ) mRNA was the highest in the PDGF-BB group, and gradually reduced in the other groups treated by PD98059 of different doses. It is concluded that expression of pERK1 protein and collagen alpha1 ( I ) mRNA was up-regulated in rat PSCs activated by PDGF-BB. Meanwhile, PD98059 could inhibit PSCs activation mediated by PDGF. It is suggested that ERK1 protein plays an important role on PSCs activation mediated by PDGF signal pathway.

[Evaluation of clinical necessity of gastrointestinal decompression after excision and anastomosis of lower digestive tract].

OBJECTIVE: To evaluate the clinical necessity of postoperative gastrointestinal decompression after operation on lower digestive tract. METHODS: Three hundred and sixty-eight patients who required excision and anastomosis of lower digestive tract were randomly divided into two groups, with or without receiving gastrointestinal decompression after operation. Clinical therapeutic efficacy and complications were compared between two groups. RESULTS: The volume of gastrointestinal suction ranged from 10 ml to 520 ml each day after operation, and was less on the first day than those on the second and the third day after operation in decompression group. There was no significant difference in the average girth between two groups before and after operation. The average girths were shorter before operation than those after operation in two groups (P< 0.001). There was no significant difference in postoperative defecation and urination time between two groups (P > 0.05). The complication rate was significantly higher in decompression group than that in non-decompression group (28% vs. 8.2%, P< 0.001). The incidence of pharyngolaryngitis was up to 23.1% in decompression group. There was no difference in hospital stay between the two groups after operation. CONCLUSION: The recovery of patients who receive excision and anastomosis of lower digestive tract will benefit from non-gastrointestinal decompression.

Tumor micrometastases in mesorectal lymph nodes and their clinical significance in patients with rectal caner.

AIM: To investigate the number, size, and status of lymph nodes within the mesorectum and to explore the prognostic significance of lymph node micrometastases in patients with rectal cancer. METHODS: Thirty-one patients with rectal cancer undergone total mesorectal excision between October 2001 and October 2002 were included. Mesorectal nodes retrieved from the resected specimens were detected with a combination of haematoxylin and eosin (HE) staining and immunohistochemistry (IHC). The relations between lymph node metastases, micrometastases and postoperative recurrence were analyzed. RESULTS: A total of 548 lymph nodes were harvested, with 17.7+/-8.2 nodes per case. The average number of metastatic nodes in HE-positive patients and micrometastatic nodes in IHC-positive patients was 5.2+/-5.1 per case and 2.2+/-1.3 per case, respectively. The mean size of all nodes and metastatic nodes was 4.1+/-1.8 mm and 5.2+/-1.7 mm in diameter, respectively. The mean size of micrometastatic nodes was 3.9+/-1.4 mm in diameter. The size of the majority of mesorectal nodes (66.8%), metastatic nodes (52.6%), and micrometastatic nodes (79.5%) was less than 5 mm in diameter. During a median follow-up period of 24.6+/-4.7 mo, 5 patients (16.7%) had recurrence, of them 2 died and 3 survived. Another case died of tumor unrelated cause and was excluded. All 5 recurrent cases had 3 or more nodes involved, and one of them developed only lymph node micrometastases. The mean number of both metastatic and micrometastatic nodes per case differed significantly between the recurrent and non-recurrent groups (P<0.01 and P = 0.01, respectively). CONCLUSION: The majority of lymph nodes, metastatic, and micrometastatic lymph nodes within the mesorectum are smaller than 5 mm in diameter. The nodal status and the number of lymph nodes involved with tumor metastases and micrometastases are related to the rapid postoperative recurrence.

Laparoscopic total mesorectal excision of low rectal cancer with preservation of anal sphincter: a report of 82 cases.

AIM: To assess the feasibility and efficacy of laparoscopic total mesorectal excision (LTME) of low rectal cancer with preservation of anal sphincter. METHODS: From June 2001 to June 2003, 82 patients with low rectal cancer underwent laparoscopic total mesorectal excision with preservation of anal sphincter. The lowest edge of tumors was below peritoneal reflection and 1.5-7 cm from the dentate line (1.5-5 cm in 48 cases, 5-7 cm in 34 cases). RESULTS: LTME with anal sphincter preservation was performed on 82 randomized patients with low rectal cancer, and 100 % sphincter preservation rate was achieved. There were 30 patients with laparoscopic low anterior resection (LLAR) at the level of the anastomosis below peritoneal reflection and 2 cm above from the dentate line; 27 patients with laparoscopic ultralow anterior resection (LULAR) at the level of anastomoses 2 cm below from the dentate line; and 25 patients with laparoscopic coloanal anastomoses (LCAA) at the level of the anastomoses at or below the dentate line. No defunctioning ileostomy was created in any case. The mean operating time was 120 minutes (ranged from 110-220 min), and the mean operative blood loss was 20 mL (ranged from 5-120 mL). Bowel function was restored and diet was resumed on day 1 or 2 after operation. The mean hospital stay was 8 days (ranged from 5-14). Postoperative analgesics were used in 45 patients. After surgery, 2 patients had urinary retention, one had anastomotic leakage, and another 2 patients had local recurrence one year later. No interoperative complication was observed. CONCLUSION: LTME with preservation of anal sphincter is a feasible, safe and minimally invasive technique with less postoperative pain and rapid recovery, and importantly, it has preserved the function of the sphincter.

[Laparoscopic total mesorectal excision for low or ultralow anterior resection of rectal cancer with anal sphincter preservation].

OBJECTIVE: To assess the feasibility of laparoscopic total mesorectal excision (TME) for low or ultralow anterior resection of rectal cancer. METHODS: Excision of the mesorectum and low (ultralow) colo-anal anastomoses were performed laparoscopically in 62 patients with low rectal cancer based on the concept of TME and double stapling technique (DST). RESULTS: Sixty-two operations with TME and DST were performed in a totally laparoscopic manner, and only one was converted to open procedures because of dysfunction of coagulation. The operative time was 125 min (110-210 min) and the operative blood loss 20 ml (5-80 ml). The time for bowel function recovery and post-operatively dietary intake was 1-2 days. Twenty-eight patients received postoperative analgesics. Average hospital stay was 8 days (5-14 days). Complications were observed in only 2 of the 62 patients, one had suffered from urinary retention and the other, anastomotic leakage. CONCLUSIONS: Totally laparoscopic excision of the mesorectum for low or ultralow anterior resection of rectal cancer is a minimally invasive technique with sphincter preservation, less postoperative pain, and rapid recovery.

[DST and low/ultralow/colo-anal anastomoses laparoscopically in the treatment of low rectal cancer].

OBJECTIVE: To assess the feasibility and adequacy of double stapling technique (DST) and anal sphincter preservation with laparoscopic approach for low rectal cancer. METHODS: DST and low/ultralow/coloanal anastomoses were performed laparoscopically on 30 patients with low rectal cancer. RESULTS: The 30 laparoscopic DST and low/ultralow/colo-anal anastomoses with anal sphincter preservation were successfully completed, and not one of the cases was converted to open procedures. The operation time was 155 min with the ranges from 110 to 320 min. The operative blood loss was 20 ml with a range between 5 and 80 ml. The time of bowel function restoration and post-operative ambulation was 1-2 days after the operation. 14 patients had postoperative analgesic requirement. The hospital stay varied from 5 to 14 days, averaging 8 days, and there were no intraoperative and postoperative complications in the 30 patients. CONCLUSION: Laparoscopic DST and low/ultralow/colo-anal anastomoses for low rectal cancer is a perspective minimally invasive technique, which is feasible, safe and effective. With the use of this technique, surgeons could accomplish higher rates of sphincter preservation, more accurate autonomic nerve preservation and good micturation with decreased postoperative pain and rapid recovery.