Maternal Treatment Of Renovascular Hypertensive Rats With Hydrogen Sulfide Inhibited Hypertension In Offspring

There is now a large body of epidemiological data providing that environmental factor acting early in life, in particular in the intrauterine and postnatal period, correlate with chronic cardiovascular diseases. Barker developed the “programming hypothesis” in 1953. Then many researches have proved that idea. Epigenetic was received more attention in environmental related diseases, and that offered us a new strategy for prevention and control of hypertension. Development of new epigenetic drugs for intervention of hypertension will become a hot topic.Hydrogen sulfide（H2S） is the third gaseous transmitter after nitric oxide and carbon monoxide. The level of H2 S in hypertension patient decreased obviously. Although H2 S was known as vasodilation factor, then a growing number of studies have found that H2 S has many biological effects, such as modulate the release of neurotransmitter and reduce the sympathetic tone and so on. Recent studies have also found that H2 S can relax human placental blood vessels, improve the blood supply of placental, and reduce the occurrence of preeclampsia. H2 S may be as a new epigenetic drug used in clinical.Renovascular hypertension（RVH） is the most common secondary hypertension, which caused by partial renal-artery stenosis always leads to persistent high blood pressure and increased activity of renin-angiotensin-aldosterone system（RAAS）, especially of angiotensin II（Ang II）. Ang II receptors subtype 1（AT1R） could be subdivided into AT1 a and AT1 b. As we all known, the activation of RAAS is involved in the pathogenesis of almost all hypertension. Renovascular hypertensive rats,therefore, is considered to be the appropriate model to study the pathogenesis of hypertension. Primary hypertension is generally believed to have a genetic predisposition. Whether secondary hypertension also has a genetic predisposition, there are few reports about these.The present study was designed to observe the effect of H2 S on the blood pressue of offspring of renovascular hypertensive rats and to elucidate the underlying mechanisms.Part I Maternal treatment of renovascular hypertensive rats with hydrogen sulfide improved baroreflex sensitivity in offspringAim: The present study was defined to observe the effect of H2 S on the blood pressue and baroreflex sensitivity of offspring of renovascular hypertensive rats and reveal the underlying mechanisms.Methods:1 Prepared rat model of renovascular hypertension7 weeks male and female Sprague-Dawley（SD） rats were used to make renovascular hypertension by 2-kidney-1-clip.2 Groups The experiment is divided into four groups. The offspring of Sprague-Dawley rats（Control）. The offspring of renovascular hypertensive rats（F1N F2N）. Maternal renovascular hypertensive rats were treated with Na HS（56μmol/Kg per day, 0.2ml/100 g, H2 S donor） during pregnancy and lactation periods and the offspring without Na HS after weaning（F1H F2N）. Maternal hypertensive rats were treated without Na HS and the offspring were treated with Na HS（56μmol/Kg per day, 0.2ml/100g） after weaning（F1N F2H）.3 Blood pressure measurement in conscious offspring Systolic blood pressure（SBP） was measured non-invasively by tail cuff plethysmography（Chengdu Instrument Factory, DSWY-1） in calm, conscious rats.4 Recording renal nerve activity Exposure renal artery by retroperitoneal and the renal sympathetic nerve was dissociated. Put the nerve on electrode to record the activity of the nerve.5 Preparation of isolated carotid sinus Preparation of isolated carotid sinus was perfused with Na HS by our previous method.6 Baroreceptor reflex sensitivity in conscious rats In order to test the barore?ex control of heart rate（HR） in conscious rats,polyethylene catheters were inserted into the femoral artery and vein for artery pressure（AP） measurement and drug injection, respectively. The catheters were tunneled subcutaneously into the back of the neck. We observed the effects of Ang II, phenylephrine（PHE）, and sodium nitroprusside（SNP） on barore?ex sensitivity in conscious rats.7 Western blot analysis of protein in nucleus tractus solitarius（NTS）We tested the protein expression of angiotensin II type 1 receptor（AT1R）,CBS, oxidative stress and inflammation related protein, such as NOX4,NLRP3 and IL-1β.Results:1 The systolic blood pressure（SBP） of offspring1.1 The SBP of male offspring The SBP of male offspring in F1 N F2N group significantly increased when compared with control group from 8 weeks to 16 weeks（respectively, 8 weeks131.9 ± 5.78 vs.113.48 ± 4.08 mm Hg, 10 weeks 133.42 ± 8.08 vs.117.42 ±5.44 mm Hg, 12 weeks 132.38 ± 4.06 vs. 116.76 ± 4.41 mm Hg, 14 weeks133.63 ± 5.29 vs. 112.45 ± 6.45 mm Hg, 16 weeks 135.22 ± 7.08 vs. 116.72 ±4.45 mm Hg, P<0.05）. Compared with male offspring in F1 N F2N group, the SBP was significantly decreased by exogenous administration of Na HS.1.2 The SBP of female offspring The SBP of female offspring in F1 N F2N group significantly increased when compared with control group from 8 weeks to 16 weeks（respectively, 8weeks 123.6 ± 3.05 vs.100.02 ± 11.88 mm Hg, 10 weeks 122.67 ± 1.98 vs.103.28 ± 9.50 mm Hg, 12 weeks 124.58 ± 2.9 vs. 105.70 ± 5.45 mm Hg, 14 weeks 119.53 ± 4.77 vs. 107.07 ± 6.46 mm Hg, 16 weeks 118.69 ± 4.89 vs.106.95 ± 7.42 mm Hg, P<0.05）. Compared with female offspring in F1 N F2N group, the above effects were significantly improved by exogenous administration of Na HS.2 The effect of H2 S on baroreflex sensitivity in anesthetized SD male rats.H2 S can increase the baroreflex sensitivity and then reduce sympathetic outflow, which may be not related to the expression of CSE in carotid sinus,may regulate the ion channels by promoting the outflow of potassium and blocking the calcium inflow in carotid sinus.3 Barore?ex sensitivity in conscious offspring3.1 Barore?ex sensitivity of male offspring Ang II induced barore?ex sensitivity appeared blunt in the male offspring of renovascular hypertensive rats from 8 weeks to 16 weeks. Even more, the baroreflex sensitivity produced by PHE was also decline. However, the baroreflex sensitivity induced by SNP was no significant difference. The above effects were reversed by treatment with Na HS.3.2Barore?ex sensitivity of female offspring Ang II induced barore?ex sensitivity appeared blunt in the female offspring of renovascular hypertensive rats from 8 weeks to 16 weeks. Even more, the baroreflex sensitivity produced by PHE was also decreased at 16 weeks. Nevertheless, the baroreflex sensitivity was induced by SNP was no significant difference. The above effects were significantly improved by exogenous administration of Na HS.4 Protein expression in NTS4.1 Protein expression in NTS of male offspring Western blot analysis showed that protein level of AT1 R and NLRP3 was elevated in male offspring of F1 N F2N group at both 8 weeks and 16 weeks.Protein expression of NOX4 and IL-1β was increased at 16 weeks in male offspring of F1 N F2N group. However, protein level of CBS was decreased in male offspring of F1 N F2N group at 16 weeks. The above effects were reversed by treatment with Na HS.1.2Protein expression in NTS of female offspring Western blot analysis showed that protein level of AT1 R and NLRP3 was elevated in female offspring of F1 N F2N group at both 8 weeks and 16 weeks.Protein expression of IL-1β was increased at 16 weeks in female offspring of F1N F2 N group. However, protein level of CBS was increased in female offspring of F1 N F2N group at 16 weeks. The protein level of NOX4 was no significant difference. The above effects were reversed by treatment with Na HS.Summary:The major findings of this part including: The SBP in the male offspring of F1 N F2N was higher than control group, even up to borderline hypertension.Although the female offspring rats in normal blood pressure range, the SBP was also higher than control group. Barore?ex sensitivity appeared blunt in the offspring of renovascular hypertensive rats that may be related to upregulation of AT1 R protein expression and the activation of oxidative stress and inflammation in NTS. Estrogen exerts protective effects by downregulation NOX4 and increasing the CBS against damage caused by Ang II, which may be the reason for the difference blood pressure between the male and female offspring. The above effects were improved by treatment with Na HS. Even more H2 S can also improve the baroreflex sensitivity and then inhibit sympathetic outflow in anesthetized male SD rat.Part II Maternal treatment of renovascular hypertensive rats with hydrogen sulfide improved autonomic nerve function in offspringObjective: The purpose of the present study was to define the effect of H2 S on the autonomic nerve function in offspring of renovascular hypertensive rats and reveal the mechanisms.Methods:1 Prepared rat model of renovascular hypertension7 weeks male or female Sprague-Dawley rats were used to make renovascular hypertension by 2-kidney-1-clip.2 Groups There are four groups in the experiment. The offspring of Sprague-Dawley rats（Control）. The offspring of renovascular hypertensive rats（F1N F2N）. Maternal renovascular hypertensive rats were treated with Na HS（56μmol/Kg per day, 0.2ml/100g） during pregnancy and lactation periods and the offspring without Na HS after weaning（F1H F2N）. Maternal hypertensive rats were treated without Na HS and the offspring were treated with Na HS after weaning（F1N F2H）.3 Investigation autonomic nervous system function In order to evaluate the cardiac autonomic tonus, we used atropine（2.5mg/kg） and propranolol（4 mg/kg） during two consecutive days, the peripheral inhibition of the sympathetic and parasympathetic activitives were induced in a reverse order.4 Western blot analysis of protein in rostral ventrolateral medulla（RVLM）We tested the protein expression of AT1 R, CBS, oxidative stress and inflammation related protein NOX4, NLRP3, IL-1β.Results:1 Autonomic nervous system function1.1 Autonomic nervous system function of male offspring The sympathetic tonus was increased in male offspring of F1 N F2N group（49.83 ± 10.52 vs. 89.4 ± 10.02 bpm P<0.05） at 8 weeks. In addition, the sympathetic effect（41.25 ± 4.78 vs. 64.5 ± 5.66 bpm P<0.05） and sympathetic tonus（63.25 ± 8.61 vs. 83.21 ± 5bpm P<0.05） after the administration of atropine and propranolol, respectively, were statistically different from those of normal SD rats at 16 weeks. Both maternal and offspring treatment with Na HS can improve the above effects.1.2 Autonomic nervous system function of female offspring The sympathetic effect after the administration of atropine and propranolol was statistically elevated（35.5 ± 3 vs. 82.00 ± 5.6bpm P<0.05） of female offspring in F1 N F2N group at 16 weeks. Both maternal and offspring treatment with Na HS can improve the above effect.2 Protein expression in RVLM2.1 Protein expression in RVLM of male offspring Western blot analysis showed that protein level of AT1 R and NOX4 was elevated in male offspring of F1 N F2N group at both 8 weeks and 16 weeks.Protein expression of NLRP3 and IL-1β was increased at 16 weeks. However,protein level of CBS was decreased in male offspring of F1 N F2N group at16 weeks. The above effects were reversed by treatment with Na HS.2.2 Protein expression in RVLM of female offspring Western blot analysis showed that protein level of AT1 R and NOX4 was elevated in female offspring of F1 N F2N group at both 8weeks and 16 weeks.Protein expression of NLRP3 and IL-1β was increased at 16 weeks. However,protein level of CBS was increased in female offspring of F1 N F2N group at16 weeks. The above effects were reversed by treatment with Na HS.Summary:The present part has found dysfunction of autonomic nervous system in offspring of renovascular hypertensive rats. The sympathetic nerve activity was increased. On the contrary, the vagus nerve activity was decreased. It may be related to upregulation of AT1 R, NOX4, NLRP3 and IL-1β protein expression in RVLM. While endogenous H2 S participated in the imbalance of autonomic nervous system. The female offspring of renovascular hypertensive rats resisted Ang II induced the sympathetic activation by increasing the production of endogenous H2 S.Part III: Maternal treatment of renovascular hypertensive rats with hydrogen sulfide increased the methylation of AT1 b geneAim: The purpose of the study is to observe the effect of H2 S on the methylation of AT1 b gene promoter.Methods:1 Prepared rat model of renovascular hypertension7 weeks male or female Sprague-Dawley rats were used to make renovascular hypertension by 2-kidney-1-clip.2 Groups The experiment included four groups. The offspring of Sprague-Dawley rats（Control）. The offspring of renovascular hypertensive rats（F1N F2N）.Maternal renovascular hypertensive rats were treated with Na HS（56μmol/Kg per day, 0.2ml/100g） during pregnancy and lactation periods and the offspring without Na HS after weaning（F1H F2N）. Maternal hypertensive rats were treated without Na HS and the offspring were treated with Na HS after weaning（F1N F2H）.3 Western blot detection of protein in kidney We tested the protein expression of AT1 R in kidney by western blot.4 Analysis the methylation of AT1 b gene promoter Genomic DNA was isolated from whole kidney using DNA QIAamp minikit（QIAgen）. DNA was treated with sodium bisulphite according to modifications of EZ DNA Methylation-Gold? Kits. Primers for amplification were designed. Gel sliced PCR products were passed through SNAP columns and used immediately for p BLUE-T-cloning according to the manufacturer’s instruction.Results:1 Protein expression of AT1 R in kidney.Compared with control group, the protein expression of AT1 R was higher in male and female offspring of F1 N F2N group at 16 weeks.2 Methylation of AT1 b promoter（NW₀47624.1 10592171-10592486） in kidney.AT1 b receptor promoter（positions, NW₀47624.1 10592171-10592486）contains 15 CG sites. Nevertheless, there was no difference between them in methylation.3 Methylation of AT1 b promoter（Genbank U01033 277-1611） in kidney.Compared with control, the methylation of AT1 b was decrease in F1 N F2N group of the first three CG sites in the position. However, the methylation increased by treatment with Na HS.4 Analysis the methylation of AT1 b by clones We prepared kidney from 16 weeks offspring for methylation analysis to focus on the above three Cp G sites in the proximal AT1 b promoter. Bisulphite converted DNA was subcloned and 10 individual clone from each animal was sequenced. This showed significantly less methylation at the three potentially methylatable sites in the proximal AT1 b promoter in F1 N F2N group（15.33% ±6.91 vs. 3.89% ± 3.89, P<0.05）.Summary:We have showed here that offspring of renovascular hypertensive rats increased expression of the AT1 receptor protein in the kidney at 16 weeks. In addition, the AT1 b gene promoter, which quite clearly is highly susceptible to regulate by DNA methylation, is indeed less methylated in programmed animals（F1N F2 N group） at 16 weeks. However, the methylation increased by treatment with Na HS.Conclusions:The major findings of this experience including: The SBP in the male offspring of F1 N F2N was higher than control group, even up to borderline hypertension. Although the female offspring rats in normal blood pressure range, the SBP was also higher than control group. Barore?ex sensitivity appeared blunt in the offspring of renovascular hypertensive rats and the sympathetic nerve activity was increased. The AT1 b gene promoter is indeed less methylated in F1 N F2N group that may be related to upregulation of AT1 R protein expression and the activation of oxidative stress and inflammation.Estrogen exerts protective effects by downregulation NOX4 and increasing the CBS against damage caused by Ang II, which may be the reason for the difference blood pressure between the male and female offspring. The above effects were improved by treatment with Na HS.