Objective To investigate the effect of endoplasmic reticulum stress on sensitivity of glioma cell line U87 to temozolomide (TMZ) and underlying mechanism.Methods (1) Glioma U87 cells were routinely cultured for 24 h in vitro;different concentrations of TMZ (0,12.5,25,50,100 μmol/L) were added to intervene the cell proliferation of U87 cells for 24 h;MTT method was used to detect the cellular proliferation inhibition rate,and half maximal inhibitory concentration (IC50) of TMZ was calculated;tunicamycin (TM) treatment (0,2,4,8 μmol/L) was given to the cells for 6 h,and then,50 μmol/L TMZ was given for 24 h,and cellular proliferation inhibition rate of U87 was detect by MTT method.(2) U87 cells were randomly divided into control group,TM group,TMZ group and TM+TMZ group;pretreatment of TM for 6 h was given to cells from TM group and TM+TMZ group;and 50 μmol/L TMZ was given to cells from TMZ group and TM+TMZ group;same amount of medium was given to cells from control group;24 h after treatment,the apoptotic rate was examined by flow cytometry;Westem blotting was used to detect the protein expressions of caspase-3,B-cell lymphoma 2 associated X protein (Bax),O-6-methlguanine-DNA methyltransferase (MGMT),glucose-regulated protein 78 (GRP78),and inositol-requiring enzyme 1 (IRE-l).Results (1) MTT showed that IC50 of TMZ was 50 μmol/L.As compared with that of 0 μmol/L TM+50 μmol/L TMZ group or 2 μmol/L TM+50 μmol/L TMZ group,the cellular proliferation inhibition rate of 4 μmol/L TM+50 μmol/L TMZ group and 8 μmol/L TM+50 μmol/L TMZ group was significantly decreased (P＜0.05).(2) As compared with TMZ group,TM+TMZ group had significantly decreased apoptotic rate (46.98％±4.79％ vs.35.74％ ±4.09％),significantly decreased caspase-3 and Bax protein expressions,and significantly increased MGMT,GRP78 and IRE-1 protein expressions (P＜0.05).Conclusion Endoplasmic reticulum stress can increase the resistance gene MGMT expression,decrease the chemotherapy sensitivity to TMZ,and induce chemoresistance ofglioma cell line U87.

Objective To investigate the effect ofeicosapentaenoic acid (EPA) on sensitivity of glioma cell line U87 to temozolomide (TMZ) and its underlying mechanism.Methods (1) U87 cells were routinely cultured in vitro,and 0,25,50,100 and 200 μmol/L EPA was given to these cells for 12,24,and 48 h;MTT assay was used to detect the cell viability.(2) U87 cells were randomly divided into control group,EPA group,TMZ group and EPA+TMZ group (concentrations of EPA and TMZ were 25 and 100 mol/L;EPA pretreatment for 12,24 and 48 h was given;TMZ was given for 24 h);MTT assay was used to evaluate the inhibition ratio of cell proliferation.(3) U87 cells were randomly divided into control group Ⅰ,EPA group Ⅰ,TMZ group Ⅰ and EPA+TMZ group Ⅰ (concentrations of EPA and TMZ were 25 and 100 mol/L;EPA pretreatment for 6 h was given;TMZ was given for 24 h);the apoptotic ratio was examined by fiow eytometry (FCM);Western blotting was used to detect the protein expressions ofcleavedcaspase-3,Bax,O-6-methlguanine-DNAmethyltransferase (MGMT),nuclear factor (NF)-κB signaling pathway related protein p65,and NF-κB inhibitor IKBα;immunofluorescent staining was employed to detect the MGMT and NF-κB p65 expressions;methylated specific PCR (MSP)was used to detect the MGMT gene promoter methylation.Results (1) The 50,100 and 200 μmol/L EPA caused concentration-dependent and time-dependent proliferation inhibition of U87 cells.(2) The inhibition ratio of cell proliferation in EPA+TMZ group was significantly higher as compared with that in the TMZ group (P＜0.05).(3) As compared with that in the TMZ group Ⅰ (34.58％±4.35％),the apoptotic ratio of U87 cells in the EPA+TMZ group Ⅰ was significantly increased (53.28％±5.05％,P＜0.05);Western blotting showed that as compared with those in TMZ group Ⅰ,the protein expressions of activated caspase-3,Bax and IKBα were significantly increased,and MGMT and NF-κB p65 protein expressions were significantly decreased in EPA+TMZ group Ⅰ (P＜0.05);immunofluorescent staining indicated that the MGMT and NF-κB p65 protein expressions in EPA+TMZ group Ⅰ were significantly lower than those in TMZ group Ⅰ (P＜0.05);the MGMT gene promoter methylaion in EPA+TMZ group Ⅰ was higher than that in TMZ group Ⅰ.Conclusion EPA enhances the sensitivity of glioma cell line U87 to TMZ,which may inhibit the MGMT expression by NF-κB dependent pathway.

Objective To investigate the effect of Eicosapentaenoic scid (EPA) on the sensitivity of glioma cell line U87 to temozolomide (TMZ) and the mechanism behind this effect.Methods U87 cells were randomly divided into four groups:control group,TMZ group,EPA+TMZ group and endoplasmic reticulum stress (ERS) activation tunicamycin group (EPA+TMZ+TM group).MTT method was used to evaluate inhibition ratio of cell proliferation.The apoptotic ratio was examined by flow eytometry.Western blot was used to detect the protein expressions of apoptosis cytokines (caspase-3 and Bax) and ERS cytokines [glucose-regulated protein 78 (GRP78) and inositol-requiring enzyme 1 (IRE-1)].Results EPA causes concentration-dependent and time-dependent inhibition of the cell proliferation (all P=0.00).EPA significantly enhanced the sensitivity of glioma cell line U87 to temozolomide.Compared to TMZ treatment alone,the inhibition ratio [(56.27+6.15)％ vs.(42.32±4.12)％,P=0.03] and apoptotic ratio [(49.78±5.94)％ vs.(37.74± 4.24)％,P=0.04] of U87 cells were enhanced by EPA+TMZ treatment.Western blot showed that the expression of apoptotic factor caspase-3 and Bax proteins were increased by EPA+TMZ treatment,while the protein expressions of ERS-related factors (GRP78 and IRE-1) were significantly inhibited (P=0.01).However,the salutary effects of EPA were reversed by ERS activation tunicamycin.Conclusion EPA enhances the sensitivity of glioma cell line U87 to temozolomide,the mechanism of which may be the suppression of ERS response.

Objective:To investigate the role of glycoprotein A repetitions predominant (GARP) in the pathogenesis of tuberculosis through regulatory T cell (Treg), in order to provide new targets for the treatment of tuberculosis.Methods:Sixty patients with active pulmonary tuberculosis (ATB) admitted to Huashan Hospital, Fudan University and Wuxi Fifth People′s Hospital from January to September 2021 were included. And six individuals with latent tuberculosis infection (LTBI), and 16 healthy controls (HC) were recruited during the same period. Flow cytometry was performed to detect the proportion of Treg in the peripheral blood, and the expressions of GARP and transforming growth factor-β1 (TGF-β1) on Treg in different groups. Mann-Whitney U test was used for statistical analysis. Results:Among the 60 patients with ATB, 23 patients did not receive anti-tuberculosis drug therapy, 17 patients were treated for less than three months, ten patients were treated for three to less than six months, and ten patients were treated for greater than or equal to six months. The percentage of CD4 + CD25 + forkhead box protein 3 (Foxp3) + Treg in untreated ATB patients was 7.50%(5.67%, 9.00%), which was higher than that in HC (5.57%(5.03%, 6.09%)), and the difference was statistically significant ( U=95.00, P=0.010). The percentage of GARP expressing in CD4 + CD25 + Foxp3 + Treg in untreated ATB patients was 10.37%(7.79%, 12.90%), which was higher than that in LTBI (7.02%(5.15%, 8.81%)) and HC (5.33%(4.26%, 6.67%)), respectively, and the differences were both statistically significant ( U=31.00, P=0.040; U=36.00, P<0.001, respectively), while there was no significant difference between LTBI and HC ( U=25.00, P=0.095). The percentage of CD4 + CD25 + Foxp3 + Treg expressing TGF-β1 in untreated ATB patients was 7.13%(4.25%, 8.89%), which was higher than that in HC (3.59%(2.10%, 5.17%)), and the difference was statistically significant ( U=71.00, P=0.001). The expressions of GARP in CD4 + CD8 -CD25 + Foxp3 + Treg in patients with ATB treated for less than three months group, three to less than six months group and greater than or equal to six months group were 7.82%(3.94%, 13.17%), 6.92%(5.61%, 9.47%) and 7.26%(5.82%, 9.64%), respectively. The expressions of TGF-β1 in CD4 + CD8 -CD25 + Foxp3 + Treg in the above three treatment groups were 11.16%(7.91%, 15.23%), 8.66%(5.43%, 12.54%) and 7.82%(6.01%, 9.53%), respectively, and the expression of TGF-β1 in CD4 + CD8 -CD25 + Foxp3 + Treg in the patients with ATB treated for less than three months group was higher than that in the greater than or equal to six months group, the difference was statistically significant ( U=37.50, P=0.024). Conclusions:Foxp3/GARP/TGF-β1 pathway may be involved in the immune mechanism of Treg regulating the pathogenesis of tuberculosis, and GARP may be a new target for anti-tuberculosis therapy.