Role Of PAR1 In Heat Stress-induced Inflammatory Responses

Objective:To study the role of protein activated receptor 1(PAR1) in heat stress-induced inflammatory responses in vascular endothelial cells, and to further clarify the role of PAR1 in systemic inflammatory response in severe heat stroke mice.Methods:1. Expressions of TNF-α, IL-6, ICAM-1 were determined respectively by RT-PCR and ELISA in HUVECs in different periods after 42℃ heat stress 2 h in vitro;2. Mononuclear cells adhesion to HUVECs was observed by Fluorescence microscopy in HUVECs in different periods after heat stress;3. Expressions of PAR1 protein and mRNA were determined by western blot and RT-PCR respectively after HS in different periods;4. Expressions of PAR1 protein were determined by Western blot after pre-treated with PAR1 siRNA and PAR1 adenovirus respectively in HUVECs in 8 h after heat stress;5. Expressions of TNF-α, IL-6, ICAM-1 mRNA were determined by RT-PCR after pre-treated with PAR1 inhibitor, agonists, siRNA and PAR1 adenovirus respectively in HUVECs in 8 h after heat stress;6. Expressions of TNF-α, IL-6, ICAM-1 in cells supernatant were determined by ELISA after pre-treated with PAR1 inhibitor, agonists, siRNA and PAR1 adenovirus respectively in HUVECs in 8 h after heat stress;7. Mononuclear cells adhesion to HUVECs was observed by Fluorescence microscopy after pre-treated with PAR1 inhibitor, agonist, siRNA and PAR1 adenovirus respectively in HUVECs in 8 h after heat stress;8. Expressions of PAR1 were determined in heart, liver, lung and small intestine in control mice and severe heat stroke by immunohistochemical assay in vivo;9. Expressions of TNF-α, IL-6, ICAM-1 in control mice and severe heatstroke mice serum were determined by ELISA after pre-treated with PAR1 inhibitor, agonist, siRNA and PAR1 adenovirus respectively in HUVECs in 6 h after heat stress;10. Influences of organs damage in severe heatstroke mice were observed after pre-treated with PAR1 inhibitor, agonist respectively;11. Influences of 72 h survival rates in severe heat stroke mice after pre-treated with PAR1 inhibitor, agonist respectively.Results:1. Expressions of TNF-α, IL-6, ICAM-1 mRNA increased after heat stress (Figure 1.1 Table 1.1);2. Expressions of TNF-α, IL-6, ICAM-1 in cells supernatant increased after HS (Table 1.2);3. Mononuclear cells adhesion to HUVECs increased after HS(Figure 3 Table 3);4. Expressions of PAR1 mRNA and protein increased in HUVECs after HS;5. PAR1 siRNA reduced expression of PAR1 protein after HS effectively, AdPARl increased expression of PAR1 protein after HS effectively (Figure 4 Table 4);6. Inhibition or knockdown of PAR1 reduced mRNA expressions of cytokines TNF-α, IL-6 and adhesion factor ICAM-1, whereas activation or over expression increased the expression of these genes (Figure 5.15.2 Table 5.15.2);7. Inhibition or knockdown of PAR1 reduced cytokines TNF-α, IL-6 and adhesion factor ICAM-1 protein secretion, whereas activation or over expression increased the protein secretion (Table 5.3 5.4);8. Inhibition of PAR1 reduced the adhesion of mononuclear leucytes to HUVECs, while the activation of PAR1 enhanced the adhesion (Figure 6.1 Table 6.1)9. Knockdown of PAR1 reduced the adhesion of mononuclear leucytes to HUVECs, whereas PAR1 over expression enhanced the adhesion (Figure 6.2 Table 6.2);10. Expression of PAR1 protein in heart, liver, lung and small intestine in severe heat stroke mice increased(Figure 7);11. Inhibition of PAR1 reduced levels of TNF-α, IL-6 and ICAM-1 in severe heat stroke mice serum, while the activation of PAR1 increased these proteins level(Table 7).12. Inhibition of PARl eased organs damage in heart, liver, lung, small intestine in severe heat stroke, while the activation of PARl will aggravated the above organs damage(Figure 8)13. Inhibition of PARl improved the survival rate of 72 h in severe heat stroke mice, while the activation of PARl reduced the survival rate of 72h in severe heat stroke mice(Figure 9, Table 9). Conclusion:1. HS can lead to PARl activation in endothelial cells, which leads to the activation of inflammatory pathways in vascular endothelial cells2. PARl is involved in the activation of systemic inflammation in severe heat stroke mice.