Objective:To discuss the enhancing effect of cordycepin on ferroptosis inducer RSL3-induced ferroptosis in the hepatocellular carcinoma HepG2 cells,and to clarify its potential mechanism.Methods:The HepG2 cells were divided into control group,RSL3 group,low,medium and high doses of cordycepin groups,RSL3+low,medium and high doses of cordycepin groups,RSL3+medium-dose cordycepin+ferroptosis inhibitor Ferrostatin-1(Fer-1)group,and RSL3+medium-dose cordycepin+ferroptosis inhibitor Liproxstatin-1(Lip-1)group.The HepG2,Huh-7 and HCCLM3 cells were treated with 0,1,5,10,15 and 20 μmol·L-1 RSL3 for 24,48 and 72 h,respectively.Cell counting kit-8(CCK-8)method was used to detect cell viability and determine the optimal concentration and treatment time of RSL3.The HepG2 cells were treated with 0,50,100,200,400,600,800,1 000,and 1 200 μmol·L-1 cordycepin for 24,48 and 72 h,respectively.CCK-8 method was used to detect the survival rate of the cells and the half maximal inhibitory concentration(IC50)was calculated to determine the optimal concentration and treatment time of cordycepin;the apoptosis inhibitor Z-VAD-FMK,autophagy inhibitor Chloroquine(CQ),necroptosis inhibitor Necrostatin-1(Nec-1),Fer-1,Lip-1,Deferasirox and 2,2,6,6-tetramethylpiper idinoxy(TEMPO)were used to treat HepG2 cells,and the survival rate of the cells was calculated;2',7'-Dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescence probe was used to detect reactive oxygen species(ROS)levels in the HepG2 cells in various groups;C11 BODIPY 581/591 fluorescence probe was used to detect lipid peroxidation(LPO)levels in the HepG2 cells in various groups;FeRhoNox-1 fluorescent probe was used to detect ferrous ion(Fe2+)levels in the HepG2 cells in various groups;kits were used to detect glutathione(GSH)and malondialdehyde(MDA)levels in the HepG2 cells;Western blotting method was used to detect the expression levels of ferroptosis-related proteins,nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)proteins in the hepG2 cells in various groups;transmission electron microscope was used to observe the ultrastructural morphology of the HepG2 cells in various groups.Results:The CCK-8 results showed that when the cells were treated with 0.56 μmol·L ?1 RSL3,the viabilities of the three cell types differed significantly.Compared with 0 μmol·L?1 RSL3 group,the survival rates of the cells in 6.4 and 12.8 μmol·L-1 RSL3 groups were significantly decreased(P<0.05).The HepG2 cells had the highest IC50 value and were selected for subsequent experiments.Compared with 0 μmol·L-1 cordycepin group,the survival rates of the HepG2 cells in 200,400,600,800,1 000,and 2 000 μmol·L-1 cordycepin groups were significantly decreased(P<0.05 or P<0.01).0.5×IC50(267.9 μmol·L-1),1×IC50(535.8 μmol·L-1)and 1.5×IC50(803.7 μmol·L-1)were selected as low,medium and high doses of cordycepin groups,respectively,with an intervention time of 24 h.Compared with control group,the survival rates of the HepG2 cells in low,medium,and high doses of cordycepin groups were significantly decreased(P<0.05).Compared with low,medium,and high doses of cordycepin groups,the survival rates of the HepG2 cells in Z-VAD-FMK+low,medium,and high doses of cordycepin groups were significantly increased(P<0.01),and those in Fer-1+medium and high doses of cordycepin groups and Lip-1+low,medium,and high doses of cordycepin groups were significantly increased(P<0.05).Compared with control group,the survival rates of the HepG2 cells in RSL3 group,RSL3+low,medium,and high doses of cordycepin groups,RSL3+cordycepin+Fer-1 group and RSL3+cordycepin+Lip-1 group were significantly decreased(P<0.05 or P<0.01).Compared with RSL3 group,the survival rates of the HepG2 cells in RSL3+low,medium,and high doses of cordycepin groups were significantly decreased(P<0.05).The DCFH-DA results showed that compared with control group,the ROS levels in the cells in medium and high doses of cordycepin groups,RSL3 group and RSL3+low,medium,and high doses of cordycepin groups were significantly increased(P<0.05 or P<0.01).The C11 BODIPY 581/591 results showed that compared with control group,the LPO levels in the HepG2 cells in medium and high doses of cordycepin groups,RSL3 group and RSL3+low,medium and high doses of cordycepin groups were significantly increased(P<0.05 or P<0.01).Compared with RSL3 group,the LPO levels in the HepG2 cells in RSL3+low,medium,and high doses of cordycepin groups were significantly increased(P<0.05 or P<0.01).The FeRhoNox-1 results showed that compared with control group,the Fe2+levels in the HepG2 cells in medium and high doses of cordycepin groups,RSL3 group and RSL3+low,medium,and high doses of cordycepin groups were significantly increased(P<0.05 or P<0.01).Compared with RSL3 group,the Fe2+levels in the HepG2 cells in RSL3+low,medium,and high doses of cordycepin groups were significantly increased(P<0.05 or P<0.01).Compared with control group,the MDA levels in the HepG2 cells in high doses of cordycepin group,RSL3 group and RSL3+low,medium,and high doses of cordycepin groups were significantly increased(P<0.05 or P<0.01).Compared with RSL3 group,the MDA levels in the HepG2 cells in RSL3+low,medium,and high doses of cordycepin groups were significantly increased(P<0.05 or P<0.01).Compared with control group,the GSH levels in the HepG2 cells in medium and high doses of cordycepin groups,RSL3 group and RSL3+low,medium,and high doses of cordycepin groups were significantly decreased(P<0.05 or P<0.01).Compared with RSL3 group,the GSH levels in the HepG2 cells in RSL3+low,medium,and high doses cordycepin groups were significantly decreased(P<0.05 or P<0.01).Compared with control group,the ultrastructure of the HepG2 cells in low and medium doses of cordycepin groups showed no significant changes,while the cells in high dose of cordycepin group exhibited reduced mitochondrial cristae,mild swelling and increased membrane density,with slightly distorted inner membrane structure.The cells in RSL3 group and RSL3+low,medium,and high doses of cordycepin groups all showed ultrastructural changes characteristic of ferroptosis.Compared with RSL3 group,the cells in RSL3+low,medium,and high doses of cordycepin groups exhibited ruptured mitochondrial membranes with increased membrane density,abnormally twisted or expanded inner membrane structures,and reduced or even disappeared mitochondrial cristae.The Western blotting results showed that compared with control group,the expression levels of FTH1 and GPX4 proteins in the HepG2 cells in medium and high doses of cordycepin groups were significantly decreased(P<0.05 or P<0.01),while the expression levels of Nrf2 and HO-1 proteins were significantly decreased(P<0.05 or P<0.01).Compared with control group,the expression levels of GPX4 protein in the HepG2 cells in low,medium and high doses of cordycepin groups,RSL3 group,and RSL3+cordycepin+Fer-1 group were significantly decreased(P<0.05).Compared with RSL3 group,the expression levels of GPX4 protein in the HepG2 cells in RSL3+low,medium,and high doses of cordycepin groups were significantly decreased(P<0.05).Conclusion:Cordycepin can significantly enhance RSL3-induced ferroptosis in the hepatocellular carcinoma HepG2 cells and down-regulate the expression of Nrf2 and HO-1 proteins in the HepG2 cells.

ObjectiveTo investigate the protective effect of cytochrome P4502D6 （CYP2D6） and cytochrome P4503A4 （CYP3A4）， key enzymes of drug metabolism in liver， on acute liver injury in water extract of Glycyrrhizae Radix et Rhizoma （WEOGRR）. MethodHealthy male Kunming mice were divided into normal group， model group， WEOGRR low-， medium- and high-dose groups （5， 10， 15 g·kg^-1·d^-1） and positive drug group （diammonium glycyrrhizinate， 75 mg·kg^-1·d^-1）， with 10 in each group. One week after preventive administration， acute liver injury model was induced by single intragastric administration of 270 mg·kg^-1 Tripterygium Glycosides tablets， and samples were collected after 18 h. The pathological changes of liver were observed by hematoxylin-eosin （HE） staining. Serum liver function indexes including alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， γ-glutamyl transpeptadase （γ-GT）， alkaline phosphatase （ALP）， and total bilirubin （TBIL） as well as the levels of oxidative stress indexes including malondialdehyde （MDA） and superoxide dismutase （SOD） in hepatocytes were determined by biochemical method. Real-time polymerase chain reaction （Real-time PCR） and Western blot were performed to detect the mRNA and protein expression levels of CYP2D6 and CYP3A4， respectively. ResultCompared with normal group， model group had significant hepatocyte swelling and inflammatory cell infiltration （P<0.01）， increased AST， ALT， γ-GT， ALP and TBIL （P<0.05）， elevated MDA and decreased SOD （P<0.01） as well as down-regulated mRNA and protein expression levels of CYP2D6 and CYP3A4 （P<0.05）. Compared with the model group， the normal group had intact liver structure without obvious abnormality， and the WEOGRR groups and positive drug group presented alleviated hepatocyte swelling and inflammatory cell infiltration （P<0.01）， reduced AST， ALT， γ-GT， ALP and TBIL （P<0.01）， lowered MDA and increased SOD （P<0.01） as well as up-regulated expression levels of CYP2D6 and CYP3A4 （P<0.01）. ConclusionThe protective effect of WEOGRR on acute liver injury induced by Tripterygium glycosides tablets may be related to reducing the contents of AST， ALT， γ-GT， ALP and TBIL in serum， inhibiting MDA and increasing the activity of SOD in liver cells， and enhancing the activities of CYP2D6 and CYP3A4， thus accelerating the metabolism of toxic substances.

Diabetic gastroparesis (DGP) is characterized by delayed gastric emptying caused by reduction of gastrointestinal motility. Its clinical manifestations include vomiting, nausea, belching, early satiety, postprandial fullness, and abdominal distention, etc. The mechanism of DGP is still not clear. Depletion of interstitial cells of Cajal, myopathy and neuropathy are considered to be the main pathogenic factors. Gastric prokinetics, gastric electric stimulation and endoscopic therapy are the main treatment options, but the long-term efficacy of these symptomatic treatment is not very satisfactory, which seriously affects patients' quality of life. This article reviewed the advances in study on pathogenesis and treatment of DGP.

Background: Studies have shown that microRNAs are closely related to the occurrence and development of tumors. As one of the most common carcinogenic microRNAs, the specific role and mechanism of miR-21 in the proliferation and migration of pancreatic cancer cells have not been fully elucidated. Aims: To investigate the effect and specific molecular mechanism of miR-21 on proliferation and migration of pancreatic cancer cells. Methods: PANC-1 and MIA PaCa-2 cells were transfected with lentiviral vectors to construct stable cell lines with overexpression and knockdown of miR-21, respectively. The transfection efficiency was verified by qRT-PCR. CCK-8 assay was used to detect cell proliferation, scratch test was used to detect cell migration ability. The expressions of Spry2 mRNA and protein were determined by qRT-PCR and Western blotting, respectively, and luciferase report assay was used to detect the regulatory effect of miR-21 on Spry2. Results: Overexpression of miR-21 significantly promoted PANC-1 cells proliferation and migration (P<0.05). Knockdown of miR-21 significantly inhibited MIA PaCa-2 cells proliferation and migration (P<0.05). Compared with controls, expressions of Spry2 mRNA and protein were significantly decreased after up-regulation of miR-21 (P<0.05), however, expressions of Spry2 mRNA and protein were significantly increased after down-regulation of miR-21 (P<0.05). Overexpression of miR-21 could inhibit luciferase activity of plasmids containing wild-type Spry2 3'-UTR sequence (P<0.05), and knockdown of miR-21 could enhance luciferase activity (P<0.05). Spry2 could reverse miR-21-mediated cell proliferation and migration (P<0.05). Conclusions: MiR-21 can affect biological function of pancreatic cancer cells by targeted regulating the expression of Spry2.

Female ; Humans ; Middle Aged ; Propofol ; therapeutic use ; Pulmonary Embolism ; complications ; Ventricular Fibrillation ; drug therapy ; etiology

A 59-year-old male patient received regularly aspirin,clopidogrel,etc. by mouth after operation for coronary angiography,balloon dilatation,and coronary stent implanting. Two months later,the patient developed multiple and superficial oral ulcer on his tongue and oral mucosa. The patient stopped clopidogrel medication by himself and other drugs were continued. The oral ulcer alleviated gradually and disappeared one month later. The oral ulcer recurred when the patient received clopidogrel for 1 months again and healed after the clopidogrel withdrawal.

A 59-year-old male patient received regularly aspirin,clopidogrel,etc. by mouth after operation for coronary angiography,balloon dilatation,and coronary stent implanting. Two months later,the patient developed multiple and superficial oral ulcer on his tongue and oral mucosa. The patient stopped clopidogrel medication by himself and other drugs were continued. The oral ulcer alleviated gradually and disappeared one month later. The oral ulcer recurred when the patient received clopidogrel for 1 months again and healed after the clopidogrel withdrawal.