Objective To investigate the role of Toll-like receptor 4(TLR4)in the regulation of homocys-teine(Hcy)-induced ferroptosis in macrophages.Methods Mouse macrophage cells RAW264.7 were cultured and divided into control group,Hcy intervention group(Hcy group),and Hcy plus ferroptosis inhibitor group(Hcy+Fer-1 group).After transfection with interference fragments,macrophages were treated with Hcy,and then divided into control group,Hcy intervention group(Hcy group),TLR4 interference negative control plus Hcy intervention group(si-NC+Hcy group),and TLR4 interference plus Hcy intervention group(si-TLR4+Hcy group).Macrophages were transfected with overexpression lentivirus and treated with Hcy,then were divided into control group,Hcy intervention group(Hcy group),a TLR4 overexpression negative control plus Hcy intervention group(OE-NC+Hcy group),and a TLR4 overexpression plus Hcy intervention group(OE-TLR4+Hcy group).After 48 hours of intervention,real-time fluorescent quantitative PCR and western blot were used to detect the expression levels of TLR4 in macrophages treated with Hcy;western blot was used to detect the expression levels of ferroptosis-related proteins ACSL4,GPX4,and FTH1 in macrophages,and ferrous ion assay kit to detect the concentration of Fe2+in macrophages;reactive oxygen species(ROS)assay kit and laser confocal microscopy were used to detect the content of intracellular reactive oxygen species.Results Compared with those in the control group,the expression level of the pro-ferroptosis protein ACSL4 was increased in the Hcy group(P<0.05),while the expression levels of anti-ferroptosis proteins GPX4 and FTH1 were decreased(P<0.05);the concentration of Fe2+was increased(P<0.05),and the content of ROS was increased.Meanwhile,the protein and mRNA expres-sion levels of TLR4 were both increased in the Hcy group(P<0.05).After macrophages were transfected with TLR4 interference fragments,compared with those in the si-NC+Hcy group,the expression levels of GPX4 and FTH1 were increased(P<0.05);the expression level of ACSL4 was decreased(P<0.05);the concentration of Fe2+was decreased(P<0.05),and the content of ROS was reduced in the si-TLR4+Hcy group.After macro-phages were transfected with TLR4 overexpression lentivirus,compared with those in the OE-NC+Hcy group,the expression levels of GPX4 and FTH1 were decreased(P<0.05),and the expression level of ACSL4 was increased(P<0.05)in the OE-TLR4+Hcy group.Conclusion Hcy induces the occurrence of ferroptosis in macrophages,and Toll-like receptor 4 has a positive feedback regulatory effect on ferroptosis in macrophages.

Objective To investigate the role of Toll-like receptor 4(TLR4)in the regulation of homocys-teine(Hcy)-induced ferroptosis in macrophages.Methods Mouse macrophage cells RAW264.7 were cultured and divided into control group,Hcy intervention group(Hcy group),and Hcy plus ferroptosis inhibitor group(Hcy+Fer-1 group).After transfection with interference fragments,macrophages were treated with Hcy,and then divided into control group,Hcy intervention group(Hcy group),TLR4 interference negative control plus Hcy intervention group(si-NC+Hcy group),and TLR4 interference plus Hcy intervention group(si-TLR4+Hcy group).Macrophages were transfected with overexpression lentivirus and treated with Hcy,then were divided into control group,Hcy intervention group(Hcy group),a TLR4 overexpression negative control plus Hcy intervention group(OE-NC+Hcy group),and a TLR4 overexpression plus Hcy intervention group(OE-TLR4+Hcy group).After 48 hours of intervention,real-time fluorescent quantitative PCR and western blot were used to detect the expression levels of TLR4 in macrophages treated with Hcy;western blot was used to detect the expression levels of ferroptosis-related proteins ACSL4,GPX4,and FTH1 in macrophages,and ferrous ion assay kit to detect the concentration of Fe2+in macrophages;reactive oxygen species(ROS)assay kit and laser confocal microscopy were used to detect the content of intracellular reactive oxygen species.Results Compared with those in the control group,the expression level of the pro-ferroptosis protein ACSL4 was increased in the Hcy group(P<0.05),while the expression levels of anti-ferroptosis proteins GPX4 and FTH1 were decreased(P<0.05);the concentration of Fe2+was increased(P<0.05),and the content of ROS was increased.Meanwhile,the protein and mRNA expres-sion levels of TLR4 were both increased in the Hcy group(P<0.05).After macrophages were transfected with TLR4 interference fragments,compared with those in the si-NC+Hcy group,the expression levels of GPX4 and FTH1 were increased(P<0.05);the expression level of ACSL4 was decreased(P<0.05);the concentration of Fe2+was decreased(P<0.05),and the content of ROS was reduced in the si-TLR4+Hcy group.After macro-phages were transfected with TLR4 overexpression lentivirus,compared with those in the OE-NC+Hcy group,the expression levels of GPX4 and FTH1 were decreased(P<0.05),and the expression level of ACSL4 was increased(P<0.05)in the OE-TLR4+Hcy group.Conclusion Hcy induces the occurrence of ferroptosis in macrophages,and Toll-like receptor 4 has a positive feedback regulatory effect on ferroptosis in macrophages.

AIM:To investigate the role of specific long noncoding RNA SLC25a6(lncSLC25a6)in homocys-teine(Hcy)-induced cuproptosis in cardiomyocytes.METHODS:Rat cardiomyocytes were cultured in vitro and divided into control group and Hcy group.After 48 h of intervention,the expression levels of cuproptosis-related proteins,ferre-doxin 1(FDX1)and heat shock protein 70(HSP70),were detected by Western blot and immunofluorescence staining.The oxidative stress state of cardiomyocytes was assessed using fluorescence staining,and the intracellular Cu2+levels were measured using a copper ion assay kit.Furthermore,the impact of Hcy on the expression of cuproptosis-related proteins in cardiomyocytes was analyzed following overexpression of lncSLC25a6.RESULTS:Compared with the control group,80 μmol/L Hcy significantly accelerated cardiomyocyte damage,with a notable underexpression of lncSLC25a6(P<0.05).Western blot results indicated that,compared with the control group,the expression level of FDX1 in the Hcy intervention group was significantly reduced(P<0.05),while the expression level of HSP70 was significantly elevated(P<0.05),and the expression level of copper ions in cardiomyocytes of the Hcy group was increased(P<0.05).Immunofluorescence staining showed a significant reduction in FDX1 fluorescence intensity and a significant increase in HSP70 fluorescence in-tensity in the Hcy group.Further overexpression of lncSLC25a6 significantly mitigated Hcy-induced cuproptosis in cardio-myocytes,resulting in elevated expression of FDX1 and reduced expression of HSP70(P<0.05).Pearson correlation analysis demonstrated that the expression level of lncSLC25a6 was negatively correlated with FDX1 protein expression(r=-0.676,P=0.046)and positively correlated with HSP70 expression(r=0.680,P=0.044).CONCLUSION:lnc-SLC25a6 significantly mitigates Hcy-induced cuproptosis in cardiomyocytes,positioning it as a potential therapeutic target for managing Hcy-induced cardiac injury.

Objective To study the role of ubiquitin-conjugating enzyme 9(UBC9)in the pyroptosis of homocysteine-induced macrophages mediated by small ubiquitin-like modifier(SUMO)modification.Methods First,the effects of homocysteine at different concentrations(0 μmol/L,50 μ.mol/L,100 μmol/L,150 μmol/L and 200 μmol/L)on the viability and pyrodeath of mouse macrophages(RAW264.7)were detected by CCK-8 and Western blot.Western blot was used to detect the expression levels of UBC9,SUMO-1,and the inflammatory cytokine IL-1β in different groups of cells.qRT-PCR was used to detect the mRNA expression of UBC9 before and after RNA interference and the expression of UBC9,pyrogen-related protein,and SUMO-1 after RNA interference.Results After stimulation with 100 μmol/L homocysteine,the effect of macrophage activity was minimal,and NLRP3 and Caspase-1 were the proteins with the most obvious increase in expression(P＜0.05).Compared with the Control group,the Hcy group's expression of IL-1β and SUMO-1 was increased(P＜0.01).Compared with the Control group,the Hcy group's UBC9 protein and mRNA levels were increased(P＜0.05).The expression of NLRP3,Caspase-1,IL-1β,UBC9,and SUMO-1 was decreased in the si-UBC9+Hcy group compared with the si-NC+Hcy group(P＜0.01).Conclusions Homocysteine induces pyroptosis in macrophages,and its mechanism of action is related to the up-regulation of UBC9 to induce SUMO modification.

BACKGROUND:Hyperhomocysteinemia is closely related to the function of islet β cells,but its specific molecular mechanism is not fully understood. OBJECTIVE:To investigate the role of N6 methyltransferase-like 3(METTL3)in homocysteine(Hcy)-induced autophagy of mouse islet β cells. METHODS:The 3rd and 4th generation mouse islet β cells were taken for the experiment.(1)Cell modeling and grouping:cells in control group were not treated with Hcy,while those in homocysteine group were treated with 100 μmol/L Hcy for 48 hours.(2)The mouse islet β-cells were transfected with the plasmids overexpressing Ad-METTL3 and si-METTL3 according to the instructions of LipofectamineTM 2000.Three different interfering fragments were designed,and the one with the best interfering efficiency was verified and screened by PCR.(3)After transfection,the cells were divided into control group,Hcy group,Ad-NC(negative control)+Hcy group,Ad-METTL3+Hcy group,si-NC(negative control)+Hcy group and si-METTL3+Hcy group.(4)qRT-PCR and western blot were used to detect the expression levels of METTL3 and autophagy-related proteins LC3Ⅱ/Ⅰ and p62 in cells.Insulin level was determined by ELISA to evaluate insulin secretion capacity of islet cells.Autophagy-related proteins and insulin level were detected after overexpression and interference with METTL3. RESULTS AND CONCLUSION:Compared with the control group,the expression level of LC3Ⅱ/Ⅰ was increased(P＜0.05),the expression of p62 was significantly reduced(P＜0.05),and the insulin secretion capacity was significantly decreased(P＜0.05)in the Hcy group.Compared with the control group,the protein and mRNA levels of METTL3 were reduced in the Hcy group(P＜0.05).After METTL3 silencing in islet β cells,Hcy further upregulated the expression of LC3Ⅱ/Ⅰ(P＜0.05),significantly dowregulated the expression of p62(P＜0.05),and increased the insulin level(P＜0.05).After overexpression of METTL3,Hcy significantly decreased the LC3Ⅱ/Ⅰ expression and increased the p62 expression in islet β cells(P＜0.05).To conclude,METTL3 is involved in the Hcy-induced autophagy regulation of islet β cells and plays a role in the regulation of insulin secretion.

OBJECTIVE To explore the expression of Maspin in invasive fungal rhinosinusitis (IFRS) and the value of Maspin in the diagnosis of IFRS. METHODS Forty two cases of fungal rhinosinusitis (FRS) were set as the experimental group, which included 12 cases of IFRS and 30 cases of noninvasive fungal rhino-sinusitis (NIFRS). At the same time, 30 cases of chronic rhino-sinusitis were set as control group. Immunohistochemistry (IHC) was used to detect the expression of Maspin. RESULTS Compared with the control group, the expression of Maspin in FRS group decreased statistically (t=-3.367, P<0.05). The IFRS group, compared with other two groups, had the lowest expression of Maspin (t=-3.390, P<0.05; t=-4.143, P<0.05). By using Maspin score of 5.70 as the cut-off point, the sensitivity and specificity for the diagnosis of IFRS was 91.7% and 88.3% respectively. CONCLUSION The expression of Maspin is very low in IFRS group. Down-regulation of Maspin expression may be a potential indicator for diagnosis of IFRS.

Objective To investigate the effect of cell proliferation and apoptosis induced by Pingyangmycin (PYM)and dexamethasone (DEX) on human umbilical vein endothelial cells (HUVEC)in vitro, so as to provide therotical evidence for treatment of aneurysm with PYMand DEX. Methods Control, PYM, DEX and PYM group were established after HUVEC were cultured for 24 hours. Cell morphology was observed by inverted microscope.The effect of cell proliferation and apoptosis were detected with CCK-8reagents and flow Cytometry. The apoptotic protein expression of caspase-3 was testedthrough Western blot. Results Descend of adherent cell density and the ascend of floating cells could be observed after treated with PYM and DEX for 24 hours. HUVEC could be inhibited effectively with concentration-dependent on PYM and DEX. The significant statistical difference of cell apoptosis rate between the group used for PYM alone and the group combined low-concentration PYM with DEX through Flow Cytometrywas found. There was significant statistical difference of apoptotic protein expression of caspase-3 through Western blot compared with the group used for PYM alone and the group combined low-concentration PYM with DEX. Conclusion PYM and DEXcould inhibitthe proliferation of HUVEC alone. The better effects could be observed combination low-concentration PYM with DEX , the mechanism of which might beapoptosis with low-concentration PYM and necrosis with high-concentration PYM.

[ABSTRACT]OBJECTIVETo observe the correlation of laryngopharyngeal reflux disease(LPRD) with anxiety and depression symptoms, and to study the treatment efficacy.METHODSThe clinical data of 98 cases with LPRD were retrospectively analyzed from March 2014 to December 2014. According to the result of the Hospital Anxiety and Depression Scale(HADS) scores, 56 cases were normal and 42 cases were abnormal. The abnormal group were randomly divided into treatment group(21 cases) and control group(21cases).The control group and normal HADS group were treated by omeprazole capsules, treatment group was given omeprazole capsules and flupenthixol melitracen tablets with psychological adjustment.RESULTS The effective rate of treatment group and control group was 66.67% and 14.29% respectively, the difference was statistically significant(χ2=11.958,P<0.05). The difference between the normal HADS group and the control group also was significant(χ2=8.843,P<0.05). The difference between the normal HADS group and the treatment group was not significant(χ2=1.372,P>0.05).CONCLUSION Laryngopharyngeal reflux in some patients exist state of anxiety or depression. Individualized treatment in patients with laryngopharyngeal reflux can improve the efficacy.

OBJECTIVE: To compare the effectiveness of vocal cord cyst excision with electronic laryngoscope (EL) and self-retaining laryngoscope (SRM). METHOD: Nightly-two patients, diagnosed as vocal cord cyst with strobolaryngoscope or electronic laryngoscope, were randomly divided into two groups. One group was 48 cases treated with electronic laryngoscope and another group was 44 cases treated with self-retaining laryngoscope. Electronic laryngoscopy examination and voice function assessments were performed to all patients, preoperatively and postoperatively at one week, three months and six months. RESULT: One failed cases under SRM, difficult exposure of glottic portion, were treated under EL. The recurrence rate of the two groups was of no statistical significance in three months after operation. Voice function assessment of the two groups was of no statistical significance at one week, three months and six months after operation. CONCLUSION The operation under electronic laryngoscope is a minimal invasive procedure to the laryngeal mucosa. Electronic laryngoscope had advantages such as clear view, accurate operation. Furthermore, it can be used for those that could not be treated under self-retaining laryngoscope.
Adult ; Aged ; Cysts ; surgery ; Female ; Humans ; Laryngeal Diseases ; surgery ; Laryngoscopy ; methods ; Male ; Middle Aged ; Minimally Invasive Surgical Procedures ; Vocal Cords ; surgery

OBJECTIVE: To explore the effective method to treat the glottic carcinoma invading the anterior commissure, we observed the difference of three kinds of operation to the anterior commissure of dogs by semiconductor laser. METHOD: Twelve dogs were divided into 3 groups at random, A: to cauterize to the thyroid cartilage's inner membrane of the anterior commissure, the right ventricular bands and vocal cord, the anterior and 1/3 of the left vocal cord; B: to cauterize part of the thyroid cartilage of the anterior commissure besides A; C: to open a window about 5 mm x 5 mm on the thyroid cartilage of the anterior commissure besides B. To take photos to observe the dogs' laryngeal wound with digital camera or electrolaryngoscope immediately, 1 week and 4 weeks, then to record the hoarseness, body weight and complications etc. RESULT: All the dogs' laser surgery were completed successfully. To observe the gross specimen, it was identical between the extent of excising and the design preoperation. One week later, the neonatal membrane covered the wound incomplete. Edematization, inflammatory reaction, pseudomembrane and the hoarseness were not heavy too. No obvious complication in group A. The neonatal membrane covered the wound incomplete. Edema, inflammatory reaction, pseudomembrane and granulation can be seen in group B. Exudation was heavy in local, erosion and infected, the hoarseness was severe. No other obvious complications in C. Different extent of adhesion could be seen at the anterior commissure in group A, B and C after 4 weeks, the laryngeal web formed, and the length of vocal cord was shorter than before. The color and luster of anterior commissure membrane was normal basically, the inflammatory reaction was not heavy in A and B groups. The anterior commissural membrane appeared the dark red chronic inflammatory reaction; the window was closed by neonatal membrane completely and had no infection in group C. To observe the gross specimen: the wound of anterior commissure was covered by prosthetic epithelia completely in three groups. The window was closed by complete membrane at the anterior commissure. The No. 1- 3 dog's conditions of hoarseness in three groups after 4 weeks: slight in A group, little severe in B and C group. The change of weight was not obvious at the pre or postoperative in group A and B. No obvious complication happened in each group postoperation. CONCLUSION The three operative methods have satisfied effects,the recovery of wound can cure well finally and no severe complications happened. It is valuable in clinic.
Animals ; Arytenoid Cartilage ; surgery ; Dogs ; Larynx ; surgery ; Laser Therapy ; methods ; Lasers, Semiconductor ; Thyroid Cartilage ; surgery ; Vocal Cords ; surgery