The phosphoinositide-3 kinase signaling is involved in neuroinflammation in hypertensive rats

Abstract

Aims: It has been demonstrated that neuroinflammation is associated with cardiovascular dysfunction. The phosphoinositide-3 kinase (PI3K) signaling in the rostral ventrolateral medulla (RVLM), a key region for sympathetic outflow, is upregulated and contributes to increased blood pressure (BP) and sympathetic outflow in hypertension. This study was designed to determine the role of the PI3K signaling in neuroinflammation in the RVLM of hypertension.

Methods: The normotensive WKY rats were performed by intracisternal infusion of lipopolysaccharide (LPS) or angiotensin II (Ang II) for inducing neuroinflammation. Elisa was used to determine the level of proinflammatory cytokines. Western blot was employed to detect the protein expression of PI3K signaling pathway. Gene silencing of PI3K p110δ subunit and overexpression of angiotensin-converting enzyme 2 (ACE2) were realized by injecting related lentivirus into the RVLM.

Results: In the spontaneously hypertensive rats (SHR), the PI3K signaling in the RVLM was upregulated compared with WKY, gene silencing of PI3K in the RVLM significantly reduced BP and renal sympathetic nerve activity (RSNA), but also decreased the levels of proinflammatory cytokines. In the WKY rats, central infusion of LPS and Ang II significantly elevated BP and RSNA, but also increased the levels of proinflammatory cytokines and PI3K signaling activation in the RVLM. These changes in the Ang II-induced hypertension were effectively prevented by gene silencing of PI3K in the RVLM. Furthermore, overexpression of ACE2 in the RVLM significantly attenuated high BP and neuroinflammation, as well as decreased the activation of PI3K signaling in hypertensive rats.

Conclusion: This study suggests that the PI3K signaling in the RVLM is involved in neuroinflammation in hypertension and plays an important role in the renin-angiotensin system-mediated changes in neuroinflammation in the RVLM.

Keywords: RVLM; ACE2; Hypertension; PI3K signaling; neuroinflammation.

Figure 1

Effect of PI3K knockdown in the RVLM on the levels of neuroinflammation and cardiovascular activity in SHR. A, Location of silencing PI3K in the RVLM. According to the rat's atlas (left), green fluorescent protein (GFP) was expressed in the RVLM area. B, Representative gel bands (top) and quantification bar graph (bottom) show the protein levels of PI3K p110δ in the RVLM in WKY and SHR after treatment with lenti‐PI3K shRNA injected into the RVLM. C, The levels of IL‐1β, IL‐6, and TNF‐α in the RVLM of WKY and SHR treated with lenti‐PI3K shRNA injections. Values are expressed as mean±SEM, n=5/group. *P<.05 vs WKY; ^# P<.05 vs lenti‐GFP. D, Time courses of systolic blood pressure (SBP) and HR in the WKY rats and SHR treated with lenti‐GFP or lenti‐PI3K‐shRNA injected into the RVLM (arrows). Values are expressed as mean±SEM, n=5/group, *P<.05 vs 0 wk, #P<.05 vs lenti‐GFP

Figure 2

Intracisternal infusion of Ang II significantly upregulated the PI3K signaling in the RVLM. Representative gel bands (top) and quantification results (bottom) of PI3K p110δ expression, and AKT phosphorylation in the RVLM of WKY rats received Ang II infusion. The results of Western blots are shown as mean±SEM, n=5/group, *P<.05 vs aCSF

Figure 3

Effects of PI3K knockdown in the RVLM on changes in cardiovascular function and proinflammatory cytokines induced by intracisternal infusion of Ang II (1 wk) in WKY rats. A: time courses of systolic blood pressure (SBP) and HR in the WKY rats treated with intracisternal infusion of Ang II in response to pretreatment with lenti‐GFP or lenti‐PI3K shRNA injected into the RVLM (arrows). Values are expressed as mean±SEM, n=5/group, *P<.05 vs 0 d, #P<.05 vs lenti‐GFP. B, the Ang II‐induced changes in IL‐1β, IL‐6, and TNF‐α in the RVLM in response to pretreatment with lenti‐PI3K shRNA injections. Values are expressed as mean±SEM, n=5/group. *P<.05 vs aCSF, ^# P<.05 vs lenti‐GFP

Figure 4

Effects of ACE2 overexpression in the RVLM on changes in cardiovascular function and proinflammatory cytokines induced by intracisternal infusion of LPS (2 wk) in WKY rats. A, Efficacy of overexpression of ACE2 in the RVLM. Bar graphs show the protein levels of ACE2 and the ratio of Ang‐(1‐7) to Ang II in the RVLM in WKY rats and SHR after treatment with lenti‐ACE2 injected into the RVLM. B, time courses of systolic blood pressure (SBP) and HR in the WKY rats treated with intracisternal infusion of LPS in response to pretreatment with lenti‐GFP or lenti‐ACE2 injected into the RVLM (arrows). Values are expressed as mean±SEM, n=5/group, *P<.05 vs 0 d, #P<.05 vs lenti‐GFP. C, the LPS‐induced changes in IL‐1β, IL‐6, and TNF‐α in the RVLM of WKY rats after pretreatment with ACE2 overexpression in the RVLM. Values are expressed as mean±SEM, n=5/group, *P<.05 vs aCSF; ^# P<.05 vs lenti‐GFP

Figure 5

Effect of ACE2 overexpression in the RVLM on neuroinflammation in SHR. A, Levels of IL‐1β, IL‐6, and TNF‐α in the RVLM in SHR 4 wk after ACE2 overexpression. Values are expressed as mean±SEM, n=5/group. *P<.05 vs WKY; ^# P<.05 vs lenti‐GFP

Figure 6

Effects of ACE2 overexpression in the RVLM on changes in PI3K signaling in the LPS‐induced hypertensive rats and SHR. Representative gel bands (top) and quantification data (bottom) of PI3K p110δ and AKT in the RVLM in the LPS‐treated WKY rats (A) and SHR (B) after pretreatment with overexpression of ACE2. Values are shown as mean±SEM, n=5/group, *P<.05 vs aCSF; ^# P<.05 vs lenti‐GFP