Objective:To observe the optimal time interval of multiple short-term hyperthermia in tongue squamous carcinoma Cal-27 cells, and investigate the role of Notch signaling pathway in hyperthermia-induced apoptosis of Cal-27 cells.Methods:Cal-27 cells were placed into a 42℃ incubator for 45 min, and the second hyperthermia was performed at 6 h,12 h, 24 h, 48 h and 72 h later, respectively. Cell proliferation capacity was detected by CCK-8 assay at 0 h, 12 h and 24 h after the end of hyperthermia. After determining the optimal time interval of hyperthermia, multiple cycles of hyperthermia were performed, and the effects of hyperthermia on the proliferation and apoptosis of Cal-27 cells were detected by CCK-8 assay and flow cytometry, respectively. The changes in the expression of Notch1, Jagged1 and hairy and enhancer of split homolog-1 (Hes1) mRNA and proteins were analyzed by real-time reverse transcription PCR (qRT-PCR) and Western blot. All data were expressed as mean±SD. Two-group comparison was performed by one-way ANOVA.Results:Hyperthermia at 42℃ for 45 min every 12 h consistently inhibited the proliferation of Cal-27 cells ( P<0.05). Compared with the control group, hyperthermia in the experiment group significantly inhibited the proliferation and induced the apoptosis of Cal-27 cells after 7 cycles of hyperthermia ( P<0.05), and the expression levels of Notch1, Jagged1 and Hes1 mRNA and proteins in the hyperthermia group were significantly down-regulated (all P<0.05). Conclusions:The optimal time interval of hyperthermia to inhibit the proliferation of tongue squamous cells carcinoma Cal-27 cells is 12 h. Hyperthermia might induce the apoptosis of Cal-27 cells by inhibiting the Notch signaling pathway.

Objectives To explore the effect and mechanism of saikosaponin D on cellular autophagy of ICCs by regulating the CaMKKβ/AMPK signaling pathway.Methods Rat primary ICCs cells were isolated and stimulated with glutamate to construct an autophagy model.The Ca2+level was detected by immunofluorescence.Primary ICCs cells were divided into control group,model group,model+saikosaponin D group,model+CaMKKβ inhibitor group,and model+saikosaponin D+CaMKKβ inhibitor group.The ultrastructure of autophagosomes was observed by transmission electron microscopy.The levels of Ghrelin and SP were detected by ELISA.The expressions of Ca2+and LC-3Ⅱ were detected by immunofluorescence.The protein expression levels of LC-3Ⅱ/Ⅰ、CaMKKβ,p-AMPK,Drp1,MFN2,IP3R and RyR were detected by Western blot.Results The fluorescence expression of LC-3Ⅱ was enhanced.Saikosaponin D reduced the levels of CaMKKβ,AMPK and MFN2(P<0.01),and increased the levels of LC-3Ⅱ/Ⅰ、IP3R,RyR,Drp1,Ghrelin and SP(P<0.01).The effect of Saikosaponin D combined with CaMKKβ inhibitor STO-609 was more significant.Conclusion Saikosaponin D can mediate Ca2+outflow through the CaMKKβ/AMPK signaling pathway,and affect the expression of excessive autophagy and gastrointestinal motility-related factors in ICCs cells.

ObjectiveTo observe the effect of Zuoguiwan on ovarian reserve in the female offspring rat model of prenatal stress （PS） and explore the mechanism based on Toll-like receptor 4/nuclear factor-κB p65 （TLR4/NF-κB p65） signaling pathway. MethodThirty-two pregnant rats were prepared and randomized into four groups （n=8）： control， model， Zuoguiwan （18.9 mg·kg^-1）， and vitamin E （1.44 mg·kg^-1）. Except the control group， the other three groups were subjected to chronic unpredictable mild stress （CUMS） from day 11 of pregnancy， and the modeling was accompanied by gavage with corresponding drugs until delivery. The PS model was evaluated by the sucrose preference test， open field test， and serum corticosterone （CORT） level. The estrous cycle was monitored and the morphological changes in the ovarian tissue were observed. The serum levels of estradiol （E₂）， luteinizing hormone （LH）， follicle-stimulating hormone （FSH）， and anti-Mullerian hormone （AMH） in the 75-day-old offspring rats were measured by enzyme-linked immunosorbent assay （ELISA） to evaluate the ovarian reserve. The ovary and uterus indices were calculated. The serum levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） were measured by enzyme-linked immunosorbent assay （ELISA）. The morphology of the ovarian tissue in the offspring on the day of birth and day 75 after birth was observed by hematoxylin-eosin staining. The transport of NF-κB p65 to the nucleus in the ovaries of the 75-day-old offspring was detected by the immunofluorescence （IF） assay. The expression of TLR4， NF-κB p65 and other related proteins in the ovarian tissue was determined by Western blot. ResultCompared with the control group， the model group showed reduced primordial follicles in the offspring on the day of birth （P<0.01） as well as disturbed estrous cycle， decreased ovary index and uterus index （P<0.01）， reduced corpus luteum， increased atretic follicles （P<0.01）， lowered serum levels of AMH and E₂ （P<0.01）， elevated serum levels of LH， FSH， IL-1β， and TNF-α （P<0.05， P<0.01）， and up-regulated protein levels of TLR4， NF-κB p65， recombinant myeloid differentiation factor 88 （MyD88）， and phosphorylated NF-κB inhibitor （p-IκBα） （P<0.01） in the 75-day-old offspring rats. Compared with the model group， Zuoguiwan and vitamin E increased the primordial follicles in the offspring on the day of birth （P<0.01）. Moreover， they resumed the estrous cycle， increased the ovary and uterine indices （P<0.05， P<0.01） and corpus luteum （P<0.01）， reduced atretic follicles （P<0.01）， elevated the serum levels of AMH and E₂ （P<0.05， P<0.01）， lowered the serum levels of LH， FSH， IL-1β， and TNF-α （P<0.05， P<0.01）， and down-regulated the expression of TLR4， NF-κB p65， MyD88， and p-IκB-α （P<0.05， P<0.01） in the 75-day-old offspring. ConclusionZuoguiwan can improve the ovarian reserve in the offspring rat model of congenital kidney deficiency by regulating the TLR4/NF-κB p65 signaling pathway.