Raf/MEK/ERK Signaling Pathway Is Involved in the Inhibition of Glioma Cell Proliferation and Invasion in the Ketogenic Microenvironment.

OBJECTIVE: A high-fat, low-carbohydrate ketogenic diet has been used to treat malignant glioma, in which the Raf/MEK/ERK signaling pathway is overactivated. However, whether the Raf/MEK/ERK signaling pathway is involved in the therapeutic effect of ketone bodies remains unknown. In this study, we investigated the effects of a major ketone body, 3-hydroxybutyric acid (3-HBA), on the proliferation and metastasis of malignant glioblastoma cells and the underlying mechanism. METHODS: Two human malignant glioblastoma cell lines (U87 and U251) were treated with different concentrations of 3-HBA with or without the Raf inhibitor PAF C-16 for 24 h. Cell proliferation, cell cycle, cell invasion, and phospholipase D1 (PLD1) activity were determined. Protein and gene expression levels of Raf/MEK/ERK signaling pathway members were examined. RESULTS: 3-HBA significantly decreased cell proliferation, invasion, and intracellular PLD1 activity in both U87 and U251 glioblastoma cell lines. 3-HBA treatment significantly increased the proportion of cells in the G1 phase and decreased the proportion of cells in S phase in U87 cells. In the U251 line, the proportion of treated cells in S phase was increased and proportion of cells in G2 was decreased. 3-HBA treatment also significantly decreased the protein expression levels of Raf, MEK, p-MEK, ERK, p-ERK, and PLD1 while increasing p53 expression; an effect that was similar to treatment with the Raf inhibitor. Co-treatment of 3-HBA with the Raf inhibitor further enhanced the effects of the 3-HBA in both cell lines. CONCLUSION: We confirmed that a ketogenic microenvironment can inhibit glioma cell proliferation and invasion by downregulating the expression of PLD1 through the Raf/MEK/ERK signaling pathway.

Benign esophageal stricture model construction and mechanism exploration.

Esophageal stricture is a debilitating condition that negatively impacts patients' quality of life after undergoing endoscopic mucosal resection (EMR). Despite its significance, this disease remains underexplored due to the lack of a stable animal model. Under direct visualization with choledochoscopy, we retrogradely damaged the esophageal mucosal layer through the gastrostomy to create a rat model of esophageal stricture. The development of histological defects in the mucosal layer was assessed over a 2-week period after model induction. Then the models were evaluated using X-ray barium radiography, Hematoxylin-Eosin, Masson's trichrome, Sirius red, and Victoria blue staining, multiphoton microscopic imaging. Additionally, the molecular mechanisms of esophageal stricture were explored by conducting RNA transcriptome sequencing, PCR, immunohistochemistry, and immunofluorescence staining. We successfully established fifteen rat models of esophageal stricture by injuring the mucosal layer. In the model group, the mucosal defect initially occurs and subsequently repaired. The epithelium was absent and was plastically remodeled by collagen during the acute inflammatory phase (Day 1), proliferation phase (Day 7), anaphase of proliferation (Day 10), and plastic remodeling phase (Day 14). We observed increased expression of COL1A1, acta2, FGF, IL-1, and TGF-ß1 pathway in the model group. We established a highly repeatable rat model of esophageal stricture, and our results suggest that the mucosal defect of the esophagus is a critical factor in esophageal stricture development, rather than damage to the muscularis layer. We identified Atp4b, cyp1a2, and gstk1 as potential targets for treating esophageal stricture, while the TGF-ß pathway was found to play an important role in its development.

Development of an immune-related prognostic biomarker for triple-negative breast cancer.

Purpose: Oncology studies employing digital dissection methodologies have provided some insight on the biological features of tumor microenvironment of Triple-negative breast cancer (TNBC), but molecular diagnostics rarely have therapeutic impact. We aimed to identify a novel prognostic biomarker to investigate immune characteristics of TNBC using transcriptomic features.Patients and Methods: We extracted whole transcriptome from breast cancer tissue of 30 TNBC patients and then used bioinformatics approaches to characterize the different immune cell contents in tumor tissue and para-cancerous tissue. We extract 2 indicators to describe the major differences in immune infiltration in the microenvironment between tumor tissue and para-cancerous tissue. We then combined the 2 indicators that represent the levels of increased and decreased infiltration in each sample to obtain the Immune Infiltration Score (IIS). Then we compared the tumor-infiltrating immune cell contents and immune infiltrating status in TNBC samples with CIBERSORT and ESTIMATE score to validate the IIS. Finally, 132 TNBC patients from the Cancer Genome Atlas program (TCGA) dataset was used to validate the predictive power of IIS.Results: 4 types of upregulated and 4 types of downregulated immune cells were identified in the tumor tissue samples of the TNBC patients. Then we developed a novel biomarker, IIS. Results showed that IIS score can clearly separate cancer and para-cancerous tissue. Using the same cutoff value of 0 in the TNBC-TCGA cohort, we show that those patients with a higher IIS had significantly higher PD-L1 expression and shorter progression-free survival time than those with a lower IIS value, indicating IIS score can be generalized to other TNBC datasets.Conclusion: we explored the immune infiltration landscape in 30 TNBC patients and provided IIS as a novel and reliable biomarker to evaluate the progression-free survival and prognosis of the TNBC patients.

[Research on network pharmacology of Acori Tatarinowii Rhizoma combined with Curcumae Radix in treating epilepsy].

The chemical constituents and action targets of Acori Tatarinowii Rhizoma and Curcumae Radix were screened by network pharmacological method,and the mechanism of the combination of Acori Tatarinowii Rhizoma and Curcumae Radix in the treatment of epilepsy was analyzed. All chemical constituents of Acori Tatarinowii Rhizoma and Curcumae Radix were retrieved by TCMSP,and their action targets were screened. Component target PPI network was constructed. Epilepsy-related genes were retrieved from PharmGkb database,and PPI networks of disease targets were drawn by Cytoscape software. Cytoscape software was used to merge the network,screen the core network,and further analyze the gene GO function and KEGG pathway enrichment,which was verified by experimental research. One hundred and five chemical constituents of Acori Tatarinowii Rhizoma and 222 chemical constituents of Curcumae Radix were retrieved. Nineteen compounds were selected as candidate compounds according to OB and DL values. Among them,4 chemical constituents of Acori Tatarinowii Rhizoma and 15 chemical constituents of Curcumae Radix were found. A total of 88 target proteins were retrieved by retrieving TCMSP data,and PPI network was constructed. Through PharmGkb database,29 epilepsy-related genes were retrieved and disease target network was established. Cytoscape software and plug-ins were used for network merging and core network screening,and 69 genes were screened out. Through GO function analysis and KEGG pathway analysis,the mechanism of anti-epilepsy is related to prolactin signaling pathway,HTLV-Ⅰ infection signaling pathway,MAPK signaling pathway and herpes simplex infection signaling pathway. Further experimental verification showed that the serum prolactin level in epileptic rats was significantly increased. The neurons in hippocampal CA1 area degenerated,necrotized and lost 24 hours after epileptic seizure,and some neuron interstitial edema occurred. The possible mechanism of compatibility of Acori Tatarinowii Rhizoma and Curcumae Radix is related to serum prolactin level,MAPK signaling pathway,HTLV-Ⅰ infection and herpes simplex infection. The analysis may be related to viral encephalitis caused by HTLV-Ⅰ virus and herpes simplex infection,which damages nerve cells and causes seizures.

[Mechanism of Acori Tatarinowii Rhizoma-Curcumae Radix treating depression based on network pharmacology].

To screen the target for the treatment of depression of Acori Tatarinowii Rhizoma and Curcumae Radix using the pharmacological method of network pharmacology, in order to define the mechanism of antidepressant effect. Pharmacological data (TCMSP) of forall of chemical constituents of Acori Tatarinowii Rhizoma and Curcumae Radix through traditional Chinese medicine system (TCMSP) was retrieved to screen the target sites, and construct the component target PPI network. PharmGkb database was retrieved for the genes associated with depression, and the disease target was mapped using the Cytoscape software. The Cytoscape software was used to merge the network and filter the core network, and further analyze the gene GO function and the KEGG pathway enrichment. There were 62 nodes and 87 connections on the target PPI network. The PPI network had 1 289 nodes and 17 714 connections. After the network merged, the component-target-disease network had 1 337 nodes and 17 801 connections. Through screening the core network, there were 63 nodes and 935 connections, which represented the complex interaction between the components and the target. Gene GO functional analysis suggested that biological processes, molecular functions and cell components were involved. Gene KEGG pathway enrichment analysis showed associations with misfolded protein, secretory hormone secretion, and apoptosis of hippocampal neurons. The possible mechanism for treating depression is the adjustment of wrong folding protein, sex hormone secretion and apoptosis of hippocampal neurons.

[Effects of Dandelion polysaccharide on IL-6/STAT3 signaling pathway in ulcerative colitis rats].

OBJECTIVE: To investigate the effects of dandelion polysaccharide on IL-6/STAT3 signaling pathway in rats with ulcerative colitis. METHODS: Forty SD rats were randomly divided into four groups (n=10):control group, model group, positive control group and dandelion polysaccharide group. The ulcerative colitis model was established by treated with 2,4,6-trinitrobenzene sulfonic acid (TNBS). The positive control group was treated with mesalazine 10 mg/kg·d and dandelion polysaccharide group was treated with dandelion polysaccharide 10 mg/kg·d. The levels of interleukin -6(IL-6), colonic myeloperoxidase (MPO) and interleukin-6 receptor (sIL-6Rα) were measured after 4 weeks of treatment. The pathological changes of colonic mucosa were observed in rats. The gene expressions of glycoprotein 130 (gp130), transcriptional activator3(STAT3) and IL-6 were detected. RESULTS: Compared with the normal control group, the level of serum IL-6 in the model group was significantly higher than that in the control group (P<0.01). Compared with the model group, the serum levels of IL-6 in the dandelion polysaccharide group and the methacetin group were significantly decreased (P<0.01). Compared with the model group, the MPO positive density of the rats in the dandelion polysaccharide group and the methacetin group was significantly lower than that in the normal group (P<0.01). Compared with the model group, the levels of sIL-6Rα and gp130 in the rats were significantly lower than those in the model group (P<0.01). Compared with the model group, the expressions of STAT3 and IL-6 mRNA in the intestinal tissue of the rats in the dandelion polysaccharide group and the methacetin group were decreased significantly. CONCLUSIONS: Dandelion polysaccharide can decrease the level of IL-6 in rats with ulcerative colitis, regulate the expression of sIL-6Rα and gp130 protein in IL-6/STAT3 pathway, and then down-regulate the expressions of STAT3 and IL-6 mRNA in intestinal tissue of rats, alleviate the colon inflammation state, protect and repair the mucosal tissue. Dandelion polysaccharide plays a role in the treatment of ulcerative colitis.

[Diversity of intestinal microflora in patients with depression after stroke].

OBJECTIVE: To investigate the changes in the population and distribution intestinal microflora and their relationship with depression in post-stroke patients. METHODS: Fecal specimens were obtained from 32 patients with post-stroke depression and 30 healthy adults for gene sequencing of 16S RNA V3 region of the intestinal microorganism using Roche/45 high-throughput sequencing platform. RESULTS: The genus and species of intestinal bacteria showed significant differences between the post-stroke patients and health adults. CONCLUSION: Significant changes in the structure of intestinal flora occur in patients with post-stroke depression.

Oxidative stress and hepatocellular injury induced by oral administration of Cr(3+) in chicken.

This study aims to investigate the oxidative stress and hepatocellular injury induced by Cr(3+) in chicken. Different doses of CrCl3 solutions (50% LD50 , 25% LD50 , and 12.5% LD50) and equivalent water were orally administered to chicken. Chicken liver samples were measured for the activities of antioxidant enzymes, the contents of glutathione, total antioxidant capacity (T-AOC), malondialdehyde (MDA), and hydrogen peroxide to indirectly evaluate the oxidative stress in chicken liver. Results indicated that the oral administration of Cr(3+) at high dose significantly increased (P < 0.05) the MDA levels after 28 days of exposure, with decreased T-AOC, glutathione, and antioxidant enzymes activities. Low and medium doses groups show that T-AOC, glutathione, and antioxidant enzymes activities increased after 14 days, then decreased gradually, but low and medium groups higher than control group, only high group lower than control group finally. These statistics and histopathological analysis suggest that high dose and long-term exposure of Cr(3+) induce oxidative stress and hepatocellular injury.

[Study on recovery and its influencing factors of ferulic acid and tetramethylpyrazine in cerebral microdialysis probe].

To establish a method for detecting microdialysis recovery of tetramethylpyrazine (TMP) and ferulic acid (FA) and investigating the influencing factors, providing the basis for further in vivo microdialysis experiments. The concentration of FA and TMP in dialysates were determined by high pressure liquid chromatography ( HPLC) and probe recovery were calculated respectively. The influence of the flow rates, medium concentration, temperature and in vivo probe stability on the recovery of FA and TMP were investigated by using concentration difference method (incremental method and decrement method). The recovery obtained by incremental method were similar to by decrement method. The in vitro recovery rate of FA and TMP decreased with the increase of 1-2.5 µL min(-1), and increased obviously with the temperature of 25-42 degrees C under the same conditions. The concentration of FA and TMP had no obvious effect on the probe recovery under the same flow rate. In addition, the recovery of TMP and FA remained stable and showed similar trends under the condition of four concentration cycles, indicating that the intra day reproducibility of the concentration difference method was good. The recovery of brain microdialysis probes in vivo 8 h maintained a relatively stable, but certain differences existed between different brain microdialysis probes, demonstrating that each probe was required for recovery correction in vivo experiment. Microdialysis sampling can be used for the local brain pharmacokinetic study of FA and TMP, and retrodialysis method can be used in probe recovery of FA and TMP in vivo.

[Preventive effect of 5-fluorouracil on post-ERCP pancreatitis].

OBJECTIVE: To investigate the preventive effect of 5-fluorouracil (5-FU) on post-ERCP (endoscopic retrograde cholangiopancreatography) hyperamy-lasemia and pancreatitis. METHODS: One hundred and sixty patients who underwent ERCP were divided at random into 2 groups: group A (n = 80) was given meglumine diatrizoate with 5-FU; and group B (n = 80) was used contrast medium without 5-FU. Blood was drawn in each group the day before, 6 hours and 24 hours following ERCP for amylase. RESULTS: Post-ERCP hyperamylasemia occurred in 9 patients in group A (11. 25%) and 45 in B (56.25%). The difference of both groups was notable. Pancreatitis ensued in 2 instances in group A (2.50%) and 8 in group B (10.00%). Their difference was significant. CONCLUSION: 5-FU added in meglumine diatrizoate is helpful to prevent post-ERCP hyperamylasemia and pancreatitis.

[New techniques of postoperative cholelith endoscopy via the "T" tube fistula].

OBJECTIVE: to explore some new techniques of postoperative cholelith endoscopy via the fistula. METHODS: Netting, washing, clamping, pushing, and electrohydraulic techniques were used in 260 cases of postoperative cholelith endoscopy via the fistula and the clinical data were analyzed retrospectively. RESULTS: The stones in 257 cases (98.8%) were removed completely in an average of 2.7 times. Three patients were operated on again because of biliary stricture and huge stones. CONCLUSION: The application of some new techniques in postoperative cholelith endoscopy via the fistula favors the complete removal of stones.