Fig 2: Expression of miR-204-5p in SH-SY5Y dopaminergic cells impairs autophagy and causes the activation of JNK-mediated apoptotic pathway. (A) Western blotting showed that expression of the miR-204-5p mimic resulted in the decrease in autophagy markers, including Beclin-1, Atg7, Atg16L1, and LC3-II/I ratio, in SH-SY5Y dopaminergic cells. (B) The protein expression of active phospho-JNKThr183/Tyr185 or phospho-c-JunSer63 was upregulated in the miR-204-5p mimic-transfected SH-SY5Y neurons. Expression of miR-204-5p led to the activation of caspase-9 and caspase-3 in SH-SY5Y dopaminergic cells. The protein expression of actin was used as the loading control and the normalization control. (C) qRT-PCR analysis showed that the mRNA level of Bim was significantly upregulated in SH-SY5Y neurons expressing miR-204-5p mimic. Each qRT-PCR experiment was performed in triplicate. (D) Expression of miR-204-5p caused a significant cell death of SH-SY5Y dopaminergic cells. For the assays of cell viability, each experiment was performed in triplicate. Each bar shows the mean ± SEM value of four independent experiments. *p < 0.05, **p < 0.01 compared with control cells.

Fig 3: A.The expression levels of proteins pathway in JNK signalling in the islet sample from indicated mice. Protein expression was examined by western blotting and normalized to the indicated total protein or GAPDH expression after 4 weeks’ HFD treatment (n = 6 samples per experimental group). B. Total and/or phosphorylated IRS1, AKT, FOXO1, and GAPDH in islet samples of HFD-fed mice that were refed for 4 h after overnight fasting and NC mice under fasting state at the end of 24 weeks. GAPDH serves as the loading control (n = 4 samples per experimental group). All values are means±s.d. Significance determined by two-way analysis of variance with general linear model procedures using a univariate approach. C. C-peptide levels in IKO and Flox mice. D. Schematic illustration of cellular events underlying Miro1-mediated insulin resistance. In response to a continuous challenge with HFD, Miro1 expression is decreased in islet beta cells leading to the activation of mtROS production and mitophagy inhibition, thereby promoting inflammatory response. The activated JNK further aggravates insulin resistance through enhancing IRS phosphorylation at Ser307 and disrupts its downstream AKT-Foxo1 phosphorylation, which contribute to inflammation and insulin resistance, and the resultant T2D.

Fig 4: Idebenone inhibited LPS-induced activation of MAPKs and NF-?B. (A) Expression levels of p-ERK, ERK, p-p38, P-38, p-JNK, and JNK were analyzed by western blotting. (B) Expression levels of p-NF-?B and NF-?B were analyzed by western blotting. (C) Idebenone inhibited LPS-caused translocation of p65 from cytosol to nucleus. BV2 microglial cells were immunostained with anti-NF-?B p65 antibodies and Alexa Fluor 594-conjugated secondary antibodies. Scale bar = 20 µm. Values are presented as the mean ± standard error. *p < 0.05, **p < 0.01, ***p < 0.001: idebenone + LPS vs. LPS; ##p < 0.01, ###p < 0.001: LPS vs. vehicle.

Fig 5: Excess ROS production contributed to nicotine induced autophagy impairment and cardiomyocytes hypertrophy through activation of p38MAPK and JNK and cilostazol decreased ROS level elevated by nicotine. (A) ROS levels were determined by using DCFH-DA probe, NAC decreased ROS significantly as positive control (Scale bar=20µm). (B) NAC pretreatment inhibited effects of nicotine on autophagy. Quantitative analysis of (C) LC3-II and (D) p62 in figure B. (E) Real-time PCR-based quantitation of cardiac hypertrophy markers. (F) Cilostazol pretreatment inhibited nicotine induced ROS accumulation (Scale bar=20 µm) (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05, n = 3).