Mitochondrial pathway mediated by reactive oxygen species involvement in α-hederin-induced apoptosis in hepatocellular carcinoma cells

AIM To investigate the antitumor activity of α-hederin in hepatocellular carcinoma(HCC) cells and its underlying mechanisms in vitro and in vivo.METHODS SMMC-7721, Hep G-2 and Huh-7 HCC cells were cultured in vitro and treated with α-hederin(0, 5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L, 25 μmol/L, 30 μmol/L, 35 μmol/L, 40 μmol/L, 45 μmol/L, 50 μmol/L, 55 μmol/L, or 60 μmol/L) for 12 h, 24 h, or 36 h, and cell viability was then detected by the Cell Counting Kit-8. SMMC-7721cells were treated with 0, 5 μmol/L, 10 μmol/L, or 20 μmol/L α-hederin for 24 h with or without DL-buthionineS,R-sulfoximine(2 mmol/L) or N-acetylcysteine(5 mmol/L) pretreatment for 2 h, and additional assays were subsequently performed. Apoptosis was observed after Hoechst staining. Glutathione(GSH) and adenosine triphosphate(ATP) levels were measured using GSH and ATP Assay Kits. Intracellular reactive oxygen species(ROS) levels were determined by measuring the oxidative conversion of 2',7'-dichlorofluorescin diacetate. Disruption of the mitochondrial membrane potential was evaluated using JC-1 staining. The protein levels of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C were detected by western blotting. The antitumor efficacy of α-hederin in vivo was evaluated in a xenograft tumor model.RESULTS The α-hederin treatment induced apoptosis of HCC cells. The apoptosis rates in the control, low-dose α-hederin(5 μmol/L), mid-dose α-hederin(10 μmol/L) and highdose α-hederin(20 μmol/L) groups were 0.90% ± 0.26%, 12% ± 2.0%, 21% ± 2.1% and 37% ± 3.8%, respectively(P < 0.05). The α-hederin treatment reduced intracellular GSH and ATP levels, induced ROS, disrupted the mitochondrial membrane potential, increased the protein levels of Bax, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C, and decreased Bcl-2 expression. The α-hederin treatment also inhibited xenograft tumor growth in vivo. CONCLUSION The α-hederin saponin induces apoptosis of HCC cells via the mitochondrial pathway mediated by increased intracellular ROS and may be an effective treatment for human HCC.

Capsule endoscopy and single-balloon enteroscopy in small bowel diseases: Competing or complementary?

AIM To evaluate diagnostic yields of capsule endoscopy(CE) and/or single-balloon enteroscopy(SBE) in patients with suspected small bowel diseases.METHODS Were trospectively analyzed 700 patients with suspected small bowel diseases from September 2010 to March 2016. CE, SBE, or SBE with prior CE was performed in 401, 353, and 47 patients, respectively. Data from clinical and endoscopy records were collected for analysis. Indications, procedure times, diagnostic yields, and complications were summarized and evaluated.RESULTS The overall diagnostic yield for the CE group was 57.6%. The diagnostic yield of CE in patients with obscure gastroin testinal bleeding(OGIB) was significantly greater than that in patients with no bleeding(70.5% vs 43.8%, P < 0.01). The overall diagnostic yield of SBE was 69.7%. There was no difference in the diagnostic yield of SBE between patients with OGIB and those with no bleeding(72.5% vs 68.9%, P = 0.534). Forty-seven patients underwent CE prior to SBE. Among them, the diagnostic yield of SBE with positive findings on prior CE was 93.3%. In addition, SBE detected two cases with superficial ulcer and erosive lesions in the small bowel, which were missed by CE. However, one case with lymphoma and two with Crohn's disease were not confirmed by SBE. The rate of capsule retention was 2.0%. There were no significant complications during or after SBE examinations.CONCLUSION SBE is a safe and effective technique for diagnosing small bowel diseases. SBE with prior CE seemed to improve the diagnostic yield of small bowel diseases.

The combination of creatine kinase-myocardial band isoenzyme and point-of-care cardiac troponin/contemporary cardiac troponin for the early diagnosis of acute myocardial infarction

BACKGROUND:The early diagnosis of acute myocardial infarction(AMI)remains challenging,especially for institutions without the high-sensitive cardiac troponin(hs-c Tn)assay.Herein,we aim to assess the value of creatine kinase-myocardial band isoenzyme(CK-MB)combined with different cardiac troponin(c Tn)assays in AMI diagnosis.METHODS:This multicenter,observational study included 3,706 patients with acute chest pain from September 1,2015,to September 30,2017.We classified the participants into three groups according to the c Tn assays:the point-of-care c Tn(POC-c Tn)group,the contemporary c Tn(c-c Tn)group,and hs-c Tn group.The diagnostic value was quantified using sensitivity and the area under the curve(AUC).RESULTS:Compared to the single POC-c Tn/c-c Tn assays,combining CK-MB and POC-c Tn/c-c Tn increased the diagnostic sensitivity of AMI(56.1%vs.63.9%,P<0.001;82.7%vs.84.3%,P=0.025).In contrast,combining CK-MB and hs-c Tn did not change the sensitivity compared with hs-c Tn alone(95.0%vs.95.0%,P>0.999).In the subgroup analysis,the sensitivity of combining CKMB and c-c Tn increased with time from symptom onset<6 h compared with c-c Tn alone(72.8%vs.75.0%,P=0.046),while the sensitivity did not increase with time from symptom onset>6 h(97.5%vs.98.3%,P=0.317).The AUC of the combination of CK-MB and POC-c Tn significantly increased compared to the single POC-c Tn assay(0.776 vs.0.750,P=0.002).The AUC of the combined CKMB and c-c Tn/hs-c Tn assays did not significantly decrease compared with that of the single c-c Tn/hs-c Tn assays within 6 h.CONCLUSIONS:The combination of CK-MB and POC-c Tn or c-c Tn may be valuable for the early diagnosis of AMI,especially when hs-c Tn is not available.

Expression and role of aryl hydrocarbon receptor in Aspergillus fumigatus keratitis

●AIM:To observe the expression and role of aryl hydrocarbon receptor(Ah R)in the immune response of mouse cornea infected with Aspergillus fumigatus(A.fumigatus).●METHODS:Murine models of A.fumigatus keratitis were established by scraping the central epithelium of mouse cornea,daubing A.fumigatus on the cornea and covering with a contact lens.The mice were randomly divided into the control group and the A.fumigatus-infected(A.F.)group for 1,3 and 5 d respectively,which corneas were daily monitored by a slit lamp microscope and the clinical scores were also recorded timely after infection.In this study,immunofluorescence staining was used to detect the expression and localization of Ah R in mouse corneas,and the m RNA and protein of Ah R were detected by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.In addition,mouse peritoneal macrophages were stimulated by A.fumigatus with or without the pretreatment of Ah R antagonist CH223191 and Ah R agonist FICZ,and the tumor necrosis factor alpha(TNF-α),inducible nitric oxide synthase(i NOS),interleukin-10(IL-10)and Arg-1 m RNA were detected by RT-PCR.●RESULTS:According to the results of the slit light photography,it was clearly indicated that the corneal inflammation were the most severe and the clinical score became the highest as well on the 3 rd day after the infection of A.fumigatus.Contrasted with the control group,the expression of Ah R in the corneal epithelial cells infected with A.fumigatus was significantly increased detected by immunofluorescence staining.Ah R mainly expressed in the nucleus and cytoplasm of corneal epithelial cells.Consistent with the transcriptional level of Ah R m RNA,the expression level of Ah R protein reached the peak on the 3 rd day after infection which was detected by Western blot.Furthermore,RT-PCR showed that CH223191 up-regulated the expression of TNF-αand i NOS and down-regulated the expression of IL-10 and Arg-1 in peritoneal macrophages;inversely,FICZ reduced the expression of TNF-αand i NOS while elevated the expression of IL-10 and Arg-1.●CONCLUSION:Ah R is involved in the pathogenesis of A.fumigatus keratitis and induced immune protection in anti-A.fumigatus immune response by inhibiting M1 and increasing M2 phenotype macrophage-related inflammatory factors.

Indoleamine 2,3-dioxygenase adjusts neutrophils recruitment and chemotaxis in Aspergillus fumigatus keratitis

AIM: To explore the effect of indoleamine 2,3-dioxygenase(IDO) on recruitment and chemotaxis function of neutrophils in Aspergillus fumigatus(A.fumigatus) keratitis.METHODS: C57BL/6 mice models of A.fumigatus keratitis were established by inoculating hyphae of A.fumigatus evenly on the corneas.The clinical scores and inflammatory cytokines expression were measured respectively on the 1^(st), 3^(th), 5^(th) day after infection.The 1-MT(1 mg/m L) was administered by gavage to exert an inhibitory effect on IDO during infection.The mice were divided into control group, 1-MT group, A.fumigatus(A.F.) group, and 1-MT+A.F.groups.The corneas were monitored by slit lamp microscopy, and recorded disease scores in 3 d after infection.Myeloperoxidase(MPO) assay was done to evaluate the neutrophils infiltration.Immunofluorescence staining was used to detect the recruitment of neutrophils in murine corneas.The m RNA of inflammatory cytokines was measured with reverse transcription-polymerase chain reaction(RT-PCR).RESULTS: The corneal inflammation and the clinical score reached the peak on the 3;day after the corneal infection.The m RNA of inflammatory cytokines of the A.F.group reached the highest on the 3;day after the infection accordingly.Meanwhile, the results of slit light photography indicated that inhibitors of IDO made inflammation more serious contrasted with the A.F.group on the 3;day.Besides, imunofluorescence staining and MPO indicated that 1-MT enhanced the recruitment, infiltration and chemotaxis of neutrophils obviously in contrast to the A.F.group.RT-PCR indicated that 1-MT increased the expression of CXCL-1, ICAM-1, IL-1β, and IL-8 significantly.CONCLUSION: IDO participates in the pathogenesis of A.fumigatus keratitis and plays an important role in inducing immune protection by inhibiting neutrophils-related inflammatory reaction and suppressing recruitment and chemotaxis of the neutrophils.

Capsaicin Regulates Mitochondrial Fission to Promote Melanoma Cell Apoptosis

Objective:Capsaicin(CPS)is a major component of the red pepper,and its anti-tumor property has been confirmed.However,the underlying mechanism of this anti-tumor effect has not been fully clarified,so we conducted this study to evaluate the role of mitochondrial fission and subsequent mitochondrial dysfunction in CPS-induced apoptosis of melanoma cells.Methods:Two melanoma cell lines and melanocytes were treated with CPS alone or in combination with ruthenium red(a transient receptor potential vanilloid 1[TRPV]antagonist),Z-VAD-FMK(a pan-caspase inhibitor),or N-acetyl-Lcysteine(an antioxidant).Cell vitality was tested using a cell counting kit-8 assay.The expression levels of related proteins were examined by Western blotting.Apoptosis,intracellular reactive oxygen species,mitochondrial membrane potential,adenosine triphosphate levels,and mitochondrial dynamics were analyzed by flow cytometry,luminometry,and confocal laser microscopy,respectively,and compared between groups.Results:CPS treatment significantly inhibited the vitality of melanoma cells(For A2058 cells:0 vs.120 mmol/L:[100.00%±0%]vs.[51.02%±6.40%],P<0.05;For WM35 cells:0 vs.120 mmol/L:[100.00%±0%]vs.[51.80%±3.45%],P<0.05)but exerted less impact on normal melanocytes.CPS promoted melanoma cell apoptosis through TRPV channels and the caspase cascade.CPS treatment then led to TRPV channel-dependent mitochondrial dysfunction with an increase in reactive oxygen species generation(For A2058 cells:CPS vs.CPS+RR:[2.34±0.30]vs.[1.34±0.12],P<0.05;For WM35 cells:CPS vs.CPS+RR:[2.25±0.25]vs.[1.65±0.13],P<0.05),dissipation of the mitochondrial membrane potential(Control vs.CPS:[1.00±0]vs.[0.61±0.08],P<0.05),and adenosine triphosphate reduction(P<0.05).In addition,reactive oxygen species generation contributed to CPS-induced melanoma cell apoptosis.Mitochondrial fission was subsequently proved to connect CPS treatment to mitochondrial dysfunction,which was also TRPV channel-dependent,thereby inducing melanoma cell apoptosis.Conclusion:Our study highlights the role of mitochondrial fission and its related mitochondrial dysfunction in mediating the pro-apoptotic effect of CPS in melanoma.These findings deepen our understanding of the mechanisms underlying the anti-tumor activity of CPS and indicate the clinical relevancy of extending the use of this agent for cancer therapy.

Analysis of potential factors contributing to refusal of invasive strategy after ST-segment elevation myocardial infarction in China

Background:Reduced application of percutaneous coronary intervention(PCI)is associated with higher mortality rates after STsegment elevation myocardial infarction(STEMI).We aimed to evaluate potential factors contributing to the refusal of PCI in STEMI patients in China.Methods:We studied 957 patients diagnosed with STEMI in the emergency departments(EDs)of six public hospitals in China.The differences in baseline characteristics and 30-day outcome were investigated between patients who refused PCI and those who underwent PCI.Multivariable logistic regression was used to evaluate the potential factors associated with refusing PCI.Results:The potential factors contributing to refusing PCI were older than 65 years(odds ratio[OR]2.66,95%confidence interval[Cl]1.56-4.52,P<0.001),low body mass index(BMI)(OR 0.91,95%Cl 0.84-0.98,P=0.013),not being married(OR 0.29,95%Cl 0.17-0.49,P<0.001),history of myocardial infarction(MI)(OR 2.59,95%Cl 1.33-5.04,P=0.005),higher heart rate(HR)(OR 1.02,95%Cl 1.01-1.03,P=0.002),cardiac shock in the ED(OR 5.03,95%Cl 1.48-17.08,P=0.010),pre-hospital delay(>12 h)(OR 3.31,95%Cl 1.83-6.02,P<0.001)and not being hospitalized in a tertiary hospital(OR 0.45,95%Cl 0.27-0.75,P=0.002).Compared to men,women were older,were less often married,had a lower BMI and were less often hospitalized in tertiary hospitals.Conclusions:Patients who were older,had lower economic or social status,and had poorer health status were more likely to refuse PCI after STEMI.There was a sex difference in the potential predictors of refusing PCI.Targeted efforts should be made to improve the acceptance of PCI among patients with STEMI in China.

Mutation of Residue Arginine^18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the α-subunit of cytochrome bsss （Cyt bsss） except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem （PS） Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSll complexes. In the present study, two arginine18 （R18） mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid （E） and glycine （G）. The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type （WT） levels and that, after irradiation at high light intensity, oxygen evolution for the PSll of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSll （Fv/Fm） of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the α-subunit of Cyt bsss and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt bss9 maintain the structure of the PSll complex and its activity, although it is not directly bound to the heme group.