Activation of the Cholinergic Anti-inflammatory Pathway Attenuated Angiotension II-Dependent Hypertension and Renal Injury

Figures

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  FIGURE 1. Timeline and flow chart of the complete set of experiments.

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  FIGURE 2. Treatment with GTS-21 attenuated AngII-induced hypertension. (A), representative pressure waves of arterial pressure. (B), statistic results of peripheral blood pressure parameters. Systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse pressure (PP), heart rate, max dP/dt and min dP/dt were similar between sham rats irrespective of vagotomy. Higher SBP, DBP, PP, max dP/dt and min dP/dt were found in the Ang group than in the Sham group. GTS-21 reduced SBP, PP, max dP/dt and min dP/dt in AngII-infused rats, while Ang and Ang + GTS group shared similar DBP. Vagotomy performed previously partly attenuated the effects of GTS-21 on peripheral blood pressure. *p < 0.05 vs. Sham group, # p < 0.05 vs. Ang group, ▲ p < 0.05 vs. Ang + GTS group (n = 6 for each group).

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  FIGURE 3. Treatment with GTS-21 improve baroreflex sensitivity in AngII-induced hypertension. The number of sequences (No. Detected Sequences) and the average gain of detected sequences (Baroreflex Gain) in AngII-infused rats were significantly lower than placebo-treated sham rats. AngII reduced LF mean coherence in the Ang group with no changes of HF mean coherence. Activating CAP by GTS-21 restore baroreflex sensitivity, showing as increased the number of sequences and average gain in the Ang + GTS group. Vagotomy significantly weakened the effects of GTS-21 on baroreflex sensitivity. *p < 0.05 vs. Sham group, # p < 0.05 vs. Ang group, ▲ p < 0.05 vs. Ang + GTS group (n = 6 for each group).

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  FIGURE 4. Treatment with GTS-21 inhibited renal fibrosis in AngII-induced hypertension. More fibrosis developed in renal tissue in the Ang group than in the Sham group. GTS-21 treatment, which activated CAP, shrank the fibrotic area. Vagotomy attenuated the effect of GTS-21 on fibrosis. *p < 0.05 vs. Sham group, # p < 0.05 vs. Ang group, ▲ p < 0.05 vs. Ang + GTS group (n = 6 for each group).

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  FIGURE 5. Western blotting demonstrated protein expression in EMT related biomarkers, pro-inflammatory factors, pro-fibrotic factors and activation of smad and NF-κB in renal tissue. GTS-21 increased the expression of E-cadherin in the Ang + GTS group. GTS-21 also reduced the EMT related biomarkers, pro-inflammatory factors and pro-fibrotic factors. There was less activation of the NF-κB and smad pathway in the Ang + GTS group than in the Ang group. Vagotomy partly inhibited the effect of GTS-21 on EMT related biomarkers, pro-inflammatory factors, pro-fibrotic factors and smad and NF-κB signal pathway. *p < 0.05 vs. Sham group, # p < 0.05 vs. Ang group, ▲ p < 0.05 vs. Ang + GTS group (n = 5 for each group).

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  FIGURE 6. Western blotting demonstrated protein expression in EMT related biomarkers, pro-inflammatory factors, pro-fibrotic factors and NF-κB signal pathway in NRK-52E cells. AngII decreased expression of cytokeratin 8 and E-cadherin and increased α-SMA and collagen-I in NRK-52E cells. GTS-21 inhibited the expression of α-SMA in the Ang group and promote the expression of cytokeratin 8 and E-cadherin. And GTS-21 inhibited AngII-induced phosphorylation of NF-κB p65, pro-inflammatory cytokines and fibrotic medium (collagen-I) which are promoted by AngII. α-Bgt blunted the effect of GTS-21 on EMT related biomarkers, pro-inflammatory cytokines, fibrotic mediums and NF-κB phosphorylation. *p < 0.05 vs. Con group, # p < 0.05 vs. Ang group, ▲ p < 0.05 vs. Ang + GTS group (n = 5 for each group).

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  FIGURE 7. Treatment with GTS-21 inhibited transwell migration and MTS proliferation in NRK-52E cells. Significantly more transwell migration (A, B) and higher MTS cell proliferation (C) of NRK-52E cells were found in the Ang treatment group than in the Con group. Activating CAP by CTS-21 inhibited AngII-promoted transwell migration and MTS cell proliferation of NRK-52E cells. α-Bgt attenuated the effect of GTS-21 on transwell migration and MTS cell proliferation. *p < 0.05 vs. Con group, # p < 0.05 vs. Ang group, ▲ p < 0.05 vs. Ang + GTS group (n = 6 for repetition).

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  FIGURE 8. Treatment with GTS-21 inhibited NF-κB activation, decreased α-SMA expression and increased E-cadherin expression in AngII-stimulated NRK-52E cells. (A) GTS-21 significantly suppressed the nuclear translocation of AngII-induced NF-κB p65, and α-Bgt attenuated the effect of GTS-21 on suppressing NF-κB activation, (B) GTS-21 significantly increased the expression of E-cadherin in Ang + GTS group, and α-Bgt attenuated the effect of GTS-21 on E-cadherin expression, (C) increased expression of α-SMA was found in NRK-52E cells after AngII treatment. GTS-21 inhibited α-SMA expression, while α-Bgt weakened this effect (n = 3 for repetition).