| Backgrounds Hypertension is a common disease, which has a serious threat to human health. Heart failure and stroke related to hypertension is associated with high level of blood pressure and sympathetic acitivity. However, its pathogenesis especially for primary hypertension is poorly understood.The central antihypertensive drugs can inhibit the sympathetic impulse distribution and reduce the release of vasoactive substances such as catecholamines mainly through activating ?2-adrenergic receptors(?2R) and/or I1-imidazoline receptors(I1R) on presynaptic or postsynaptic membrane within the central nervous system, which plays a hypotensive and heart rate slowing effect. But its antihypertensive mechanism is still not fully understood.It is well known that rostral ventrolateral medulla(RVLM) is the main active region of central antihypertensive drugs and it containing presympathetic neurons play a key role as the target cells in central antihypertensive effects. The dysfunction of RVLM has a close relationship with the formation and development of hypertention. Recent studies have demonstrated that increased excitability of neurons in RVLM is the main mechanism of sympathetic overactivity and inhibition of presympathetic neurons activity can effectively reduce sympathetic outflow and lower blood pressure. The presympathetic neurons in the RVLM project directly to sympathetic preganglionic neurons in the spinal cord and are recognized as a major target responsible for the action of centrally acting antihypertensive agents such as clonidine and moxonidine. Numerous studies have shown that the renin-angiotensin system(RAS)has a very closely relationship with the occurrence, development and prognosis of hypertension. In addition to the peripheral RAS, there is also a set of RAS in the central nervous system(CNS) independent of the peripheral RAS and participates in regulation of cardiovascular activity by reducing sympathetic activity and enhancing baroreflex function. However, recent studies have confirmed that angiotention II in RVLM could induce oxidative stress mediated by AT1 R, furthermore, the enhancement of oxidative stress in RVLM can cause increased neuronal excitability leading to sympathetic hyperactivity and high blood pressure. Therefore, our concerns are whether antihypertensive drugs can reduce the level of oxidative stress within the RVLM, and whether AT1 R is involved in this central enhanced oxidative stress mechanism, which is worth to study. Methods Spontaneously hypertensive rats(SHR) and normotensive WKY rats were performed In this study. Moxo and its receptor blockers(I1R?a2AR) were given to the rats via fourth ventricle catheterization and perfusion technique for 2 weeks, the animal's blood pressure, heart rate, 24-h urinal excretion of NE, and oxidative stress within the RVLM were observed; DHE staining techniques were used to detect changes in the superoxide anion content in the RVLM. Western Blot and RT-q PCR analysis were used to detect the changes in the amount of AT1 R, NADPH oxidase, and SOD expression, in order to observe the relationship between Moxo's centrally antihypertensive action and level of oxidative stress in the RVLM. Results 1. Antihypertensive effect of Moxo in RVLM was mediated by central I1 R in SHR. 1.1 Effects of chronic perfusion of Moxo into the fourth ventricle(icv) on BP and HR.After perfusion of Moxo and corresponding blockers for 2 weeks, mean arterial pressure(MAP) and HR of Moxo group(MAP: 143 ± 3mm Hg, HR: 348 ± 9bpm) and Moxo + Yoh(a2AR blocker) group(MAP: 145 ± 4 mm Hg, HR: 363 ± 5bpm) were significantly(p<0.05) lower than a CSF group(MAP: 186 ± 5mm Hg, HR: 392 ± 5bpm); and both of them were also significantly(p<0.05) lower than Moxo + Efa(I1R and a2 AR mixed blocker) group(MAP: 175 ± 3mm Hg, HR: 388 ± 4bpm). 1.2 Effects of chronic perfusion of Moxo into the fourth ventricle on RSNA.After perfusion of Moxo and corresponding blockers for 2 weeks, excretion of NE in 24-h urine of Moxo group(0.287 ± 0.055 ?g/24h) and Moxo + Yoh group(0.260 ± 0.049 ?g/24h) were significantly(p<0.05) lower than a CSF group(0.553 ± 0.047 ?g/24h), and both of them were also significantly(p<0.05) lower than Moxo + Efa group(0.489 ± 0.033 ?g/24h). 2. Effects of Moxo-mediated depression of oxidative stress within the RVLM on cardiovascular activity.After perfusion of Moxo and corresponding blockers for 2 weeks, compared with the control group which was perfused of a CSF into the fourth ventricle(icv), the group of Tempol(an analogon of SOD) microinjection has significantly(p<0.05) lower values in based MAP(-10 ± 3 VS-35 ± 2 ?mm Hg), HR(-16 ± 3 VS-40 ± 4 ?bpm) and RSNA(-6 ± 3 VS-22 ± 3 ?%). 3. Moxo reduced production of ·O2- in RVLM mediated by I1 R. 3.1After chronic perfusion(icv)of SHR and WKY rats for 2 weeks, DHE staining showed that the content of·O2- within RVLM of SHR group(33.60 ± 0.91) was increased significantly(p<0.05) compared with that of WKY group(18.40 ± 0.76). 3.2 After chronic perfusion of Moxo and corresponding blockers for 2 weeks, DHE staining showed that the content of·O2- within the RVLM of Moxo group(21.62 ± 2.67) and Moxo + Yoh group(22.39 ± 1.31) were significantly(p<0.05) lower than that of a CSF group, and both of them were also significantly(p<0.05) lower than that of Moxo + Efa group(32.28 ± 0.94). 4. Moxo downregulated the protein expression of AT1 R, NADPH oxidase(Nox4) in the RVLM, but did not affact the SOD protein expression.Western Blot suggested that the AT1 R, Nox4 protein expression in the RVLM of Moxo group and Moxo +Yoh group were significantly(p<0.05) lower than that of a CSF group, and both of them were significantly(p<0.05) lower than that of Moxo + Efa. The SOD protein expression has no difference among the above groups. 5. Effect of chronic fourth ventricle perfusion of moxonidine on the Cardiovascular responses to microinjection of Ang II in RVLM.The result shows representative original traces of the BP, HR and RSNA responses to RVLM-microinjected Ang II(10 pmol) after chronic perfusion with moxonidine for two weeks. Unilateral injection of Ang II in SHR can produce a transient pressor effect in BP(17.5±1.89 ?mm Hg), HR(8.77±2.64 ?bpm) and RSNA(20.54±3 ?%). However, chronic perfusion of moxonidine can decrease the pressor effect induced by Ang II(BP: 7.16±0.44 ?mm Hg, HR: 1.5±1.58 ?bpm, RSNA: 11±1.9 ?%). 6. Effects of moxonidine on PI3K/Akt signal pathway in RVLM.Western blot analysis revealed that chronic fourth ventricle perfusion of moxonidine can significantly decrease the protein expression of PI3 K and the phosphorylation of Akt in RVLM(P<0.05). Realtime-PCR analysis showed that the gene expression of NF-?B subunit p65 was significantly(p<0.05) downregulated after chronic treatment of Moxonidine, while the I?B(the endogenous inhibitor of NF?B) gene expression was significantly(p<0.05) increased. Western Blot also displayed that phosphorylation of p65 subunit within RVLM of Moxonidine group was significantly(p<0.05) reduced compared with a CSF group. 7. Centrally antihypertensive effect of Moxo is blunted after PI3 K si RNA infecting in the RVLM of SHR.PI3K Knock Down si RNA interference lentivirus( Len Vi-PI3KKD) or scramble lentivirus(Len Vi-GFP) were used for locally infecting the RVLM, 3 weeks after lentiviral infection and chronic perfusion of Moxo(icv) for 2 weeks, the animals' MAP and HR were observed. The results showed that after RVLM Len Vi-GFP local infection, Moxo significantly(p<0.05) reduced blood pressure compared with the vehicle control group. However, after Len Vi-PI3 K local infection, Moxo's antihypertensive effect is significantly(p>0.05) reduced compared with the vehicle control group. 8. PI3 K signal pathway contributes to moxonidine decreased the protein expression of AT1 receptors?NADPH oxidase(Nox4).Western blot analysis revealed that there were no differences among the Len Vi-PI3 K group, Len Vi-GFP+ Moxonidine group and Len Vi-PI3K+ Moxonidine group in protein expression of AT1R?Nox4(p>0.05). 9. PI3 K signal pathway contributes to moxonidine decreased the protein expression of p-p65 and increased p-I?B.Western blot analysis revealed that there were no differences among the Len Vi-PI3 K group, Len Vi-GFP+ Moxonidine group and Len Vi-PI3K+ Moxonidine group in protein expression of p-p65 and p-I?B(p>0.05). Conclusions Our findings suggest that centrally antihypertensive effect of moxonidine is resulted from decreased oxidative stress in the RVLM of hypertension, which may be dependent on the PI3K/Akt/NF?B/AT1 R signaling pathway. |
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