Critical care medicine journal

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Band density was analyzed with ImageJ and normalized to GAPDH. After stimulation, supernatant samples of cultured cell were collected. ASC speck images were acquired using a TSC SP8 confocal microscope (Leica).

ASC speck-positive microglial cells were counted using ImageJ software. The fluorescence intensity was measured using flow cytometry (CytoFLEX, Beckman) and analyzed with FlowJo. Statistical analyses were performed using GraphPad Prism 8. Melatonin ameliorated weight loss induced by MPTP treatment (Figure 1B). We next performed tyrosine hydroxylase (TH) immunostaining and Western blotting to examine the protective effect of melatonin on dopaminergic neurons.

The results indicated that compared with the control group, MPTP-treated mice exhibited severe loss of TH-positive neurons, and melatonin treatment partially alleviated this critical care medicine journal (Figure 2A and B). Furthermore, while IBA-1 positive cells in the striatum were significantly increased in the MPTP-treated mice, melatonin reduced IBA-1 expression in this region (Figure 2F and G).

Figure 2 Protective effect of melatonin on dopaminergic neurons and microglia. Yellow dotted circles indicate the SNc. Scale bars are indicated. Data are shown as representative plots (C) and bands quantified by densitometric analysis (D and E).

DAPI represents nuclear staining (blue). Data are shown as representative plots (H) and bands quantified by densitometric analysis (I and J). However, melatonin markedly inhibited the activation of the NLRP3 inflammasome by decreasing the levels of NLRP3 and cleaved-caspase 1 in the SN and the striatum (Figure 3A and B, E and F, G and H, K and L).

These results suggest that melatonin prevents MPTP-induced NLRP3 inflammasome activation in vivo. Figure 3 Melatonin inhibits NLRP3 inflammasome activation in vivo. Representative blots are shown critical care medicine journal (A).

In addition, we measured the intracellular production of ROS in BV2 cells to evaluate whether melatonin regulated the oxidative stress, which is an important contributor in the activation of NLRP3 inflammasome. Mean fluorescence intensity was quantified by FlowJo in (G). DAPI represents the nuclear signal (blue). White arrows indicate ASC specks. We found that melatonin significantly increased the expression of SIRT1 over time, but high concentrations of melatonin decreased SIRT1 expression levels, indicating that melatonin exerts its effects within a particular concentration range (Figure 5A and B).

Then, we evaluated the regulatory effect of melatonin on SIRT1 expression and NLRP3 inflammasome suppression. Finally, we investigated whether SIRT1 suppression affects the inhibitory effect of melatonin on NLRP3 inflammasome activation using selisistat, a specific SIRT1 inhibitor.

This suggested that melatonin negatively regulates the NLRP3 inflammasome through its effects on SIRT1 expression (Figure 5G and H). Figure 5 Inhibition of SIRT1 reverses critical care medicine journal suppressive effect of melatonin on NLRP3 inflammasome activation.

Speed drug Western blotting of SIRT1 expression is shown as plots (A) and quantified bands (B). Representative critical care medicine journal are shown in (C). Bands were quantified by densitometric analysis in (D). Specifically, MPTP pregnant maximus induced NLRP3 inflammasome activation in vivo, but only acted as a priming signal in vitro.

Melatonin ameliorated neuroinflammation in PD models by inhibiting NLRP3 inflammasome activation in vitro and in vivo. Melatonin inhibited NLRP3 inflammasome activation through a SIRT1-dependent pathway (Figure 6).

Collectively, our results demonstrated a previously unrecognized mechanism through which melatonin suppresses inflammasome-induced neuroinflammation in PD. The NLRP3 inflammasome is assembled and activated in microglia when MPTP acts as critical care medicine journal priming signal (signal 1), and ATP or nigericin acts as the activation signal (signal 2).

Although the precise pathogenesis of PD remains elusive, accumulating evidence indicates that inflammasome-induced neuroinflammation is an important component critical care medicine journal PD etiopathogenesis. Activation signals are responsible for assembly and activation of the NLRP3 inflammasome. Emerging evidence suggests that melatonin treatment protects dopaminergic neurons in PD by decreasing neuroinflammation. Here, we demonstrated that melatonin almost fake counteracted MPTP-induced NLRP3 inflammasome activation both in vivo and in vitro.

The anti-inflammatory effects of melatonin are known to be mediated by SIRT1 in some critical care medicine journal.



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