Fig 1.
(A) Schematic representation of TC parameter settings. (B) Measurement of the cell temperature during the TC treatment by the thermocouple. (C) Image of the TC controller setup.
Fig 2.
Effect of TC-HT on H2O2-induced cytotoxicity in SH-SY5Y cells.
(A) Dose-response curve of SH-SY5Y cells treated with different concentrations of H2O2 for 24 h. (B) SH-SY5Y cells were pretreated at 41.5°C temperature setting and challenged with or without 450 μM H2O2. The cell viability was measured by MTT assay at 24 h after the H2O2 treatment. (C) SH-SY5Y cells were pretreated at 42.5°C temperature setting with different thermal dosages and challenged with or without 450 μM H2O2. The cell viability was measured by MTT assay at 24h after the H2O2 treatment. (D) Comparison of the neuroprotective effect under different low temperature period settings. (E) The LDH release was measured to confirm the neuroprotective effect of TC treatment. SH-SY5Y cells were pretreated at 42.5°C temperature setting and challenged with or without 450 μM H2O2. The LDH release was measured 24h after the H2O2 treatment. Data represent the mean ± standard deviation (n = 3). ***P < 0.001 and **P < 0.01.
Fig 3.
Effect of TC-HT on Aβ-induced cytotoxicity in SH-SY5Y cells.
(A) The cell viability of SH-SY5Y cells treated with 25 or 50 μM Aβ for 4 days. (B) The TC or HT pretreatment and post-treatment at 42.5°C temperature setting were applied to the cells before or after 50 μM Aβ administration, and the cell viability was measured by MTT assay 4 days after treatment. The TC or HT treatment was applied 4 h before the Aβ administration for the pretreatment group and 1 h after Aβ administration for the post-treatment group. (C) Representative light microscopy images of SH-SY5Y cells after treatment. The integrity of the cells was destructed by Aβ, and the TC post-treatment caused the protective effect and retained the cell morphology. Scale bar = 100 μm. Data represent the mean ± standard deviation (n = 3). ***P < 0.001.
Fig 4.
Effect of TC-HT on H2O2-induced ROS generation in SH-SY5Y cells.
(A) ROS level was measured 24 h after the H2O2 treatment by flow cytometry with DHE fluorescent dye. (B) Quantification of the ROS levels after H2O2, TC+H2O2, or HT+H2O2 treatment. Data represent the mean ± standard deviation (n = 3). **P < 0.01.
Fig 5.
Effect of TC-HT on H2O2-induced MMP reduction in SH-SY5Y cells.
(A) MMP was analyzed 24 h after the H2O2 treatment by flow cytometry with DiOC6(3) fluorescent dye. (B) Quantification of the cells with decreased MMP after H2O2, TC+H2O2, or HT+H2O2 treatment. Data represent the mean ± standard deviation (n = 3). ***P < 0.001 and **P < 0.01.
Fig 6.
Effect of TC-HT on expressions of HSPs in SH-SY5Y cells.
(A) Cells were lysed 16 h after H2O2 treatment and western blot analyses of HSP70 and HSP105 expressions were performed. (B) Quantification of HSP70 and HSP105 expressions after H2O2, TC+H2O2, or HT+H2O2 treatment. The expression levels were normalized to GAPDH. Data represent the mean ± standard deviation (n = 3). ***P < 0.001.
Fig 7.
Effect of TC-HT on expressions of IDE and proteasome in SH-SY5Y cells.
(A) Cells were lysed 15 h after H2O2 treatment and western blot analyses of IDE and proteasome subunits (PSMC3 and PSMA3) expressions were performed. (B) Quantification of IDE, PSMC3, and PSMA3 expressions after H2O2, TC+H2O2, or HT+H2O2 treatment. The expression levels were normalized to GAPDH. Data represent the mean ± standard deviation (n = 3). ***P < 0.001 and **P < 0.01.
Fig 8.
Effect of TC-HT on Akt/Nrf2 and Akt/CREB signalling pathways and related protein expressions.
Cells were lysed 15 h after H2O2 treatment for p-Akt, Akt, Nrf2, p-CREB proteins, and lysed 18 h after H2O2 treatment for HO-1 protein, and western blot analyses of p-Akt, Akt, Nrf2, p-CREB, and HO-1 proteins were performed. Quantification of p-Akt, Nrf2, p-CREB, HO-1 expressions after H2O2, TC+H2O2, or HT+H2O2 treatment. The expression level of p-Akt was normalized to total Akt while other proteins were normalized to GAPDH. Data represent the mean ± standard deviation (n = 3). ***P < 0.001, **P < 0.01 and *P < 0.05.
Fig 9.
Effect of LY294002 on the neuroprotective effect of TC-HT and related protein expressions.
(A) TC treatment conferred neuroprotective effect and significantly increased the cell viability of SH-SY5Y neuron cells under the H2O2 stress. The neuroprotective effect of TC treatment was abrogated by addition of the PI3K inhibitor LY294002 in a dose-dependent manner. (B) Cells were lysed 15 h after H2O2 or 25 μM LY294002 treatment, and western blot analyses of p-Akt and Nrf2 proteins were performed. The inhibitor LY294002 reversed the activated levels of p-Akt and Nrf2 induced by TC treatment. The expression level of p-Akt and Nrf2 was normalized to total Akt and GAPDH, respectively. We used the abbreviation “LY” to represent the PI3K inhibitor LY294002 in the figure. Data represent the mean ± standard deviation (n = 3). ***P < 0.001, **P < 0.01 and *P < 0.05.
Fig 10.
The proposed mechanisms for the protective effect of TC-HT against H2O2 or Aβ-induced neural injury.
The epidermal growth factor receptor (EGFR) in the cell membrane could be the upstream receptor sensing extracellular TC-HT stimulation and transmit the signal to activate the PI3K/Akt pathway, which induces Nrf2 and CREB activations. The transcription factors entering the nucleus further enhance the expressions of HO-1 and other prosurvival proteins which decrease the ROS level and inhibit the apoptosis signal.