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Sustained elevation of NF-κB activity sensitizes offspring of maternal inflammation to hypertension via impairing PGC-1α recovery

Growing evidence has demonstrated that maternal detrimental factors, including inflammation, contribute to the development of hypertension in the offspring. The current study found that offspring subjected to prenatal exposure of inflammation by lipopolysaccharide (LPS) challenge during the second semester showed significantly increased systolic blood pressure. In addition, these offspring also displayed augmented vascular damage and reactive oxygen species (ROS) levels in thoracic aortas when challenged with deoxycorticosterone acetate and high-salt diet (DOCA-salt). Interestingly, the antioxidant N-acetyl-L-cysteine markedly reversed these changes. Mechanistically, prenatal LPS exposure led to pre-existing elevated peroxisome proliferators-activated receptor-γ co-activator (PGC)-1α, a critical master of ROS metabolism, which up-regulated the ROS defense capacity and maintained the balance of ROS generation and elimination under resting state. However, continued elevation of NF-κB activity significantly suppressed the rapid recovery of PGC-1α expression response to DOCA-salt challenge in offspring that underwent prenatal inflammatory stimulation. This was further confirmed by using a NF-κB inhibitor (N-p-Tosyl-L-phenylalanine chloromethyl ketone) that restored PGC-1α recovery and prevented blood pressure elevation induced by DOCA-salt. Our results suggest that maternal inflammation programmed proneness to NF-κB over-activation which impaired PGC-1α-mediated anti-oxidant capacity resulting in the increased sensitivity of offspring to hypertensive damage.


Maternal inflammation aggravates vascular damage and hypertension in adult offspring after DOCA-salt treatment.
Recent findings have demonstrated that prenatal exposure to detrimental factors sensitizes offspring to hypertension when challenging with high-salt diet 12 or angiotensin II 20 . We first exploited the use of DOCA, an aldosterone analogue that induces the animals to a state of hypertension when combined with unilateral nephrectomy in rats 21 , as a "second hit" to evaluate the hypertensive sensitivity in adult offspring of LPS-treated mothers. At the age of 16 weeks, both control and offspring of prenatal LPS exposure received 1% NaCl in drinking water together with subcutaneous injection of DOCA every other day for 4 weeks, defined as Con+ DOCA and LPS+ DOCA group, respectively. Control and offspring of prenatal LPS exposure administrated with 50% glycerol subcutaneously alone were taken as vehicle control, defined as Con+ Ve and LPS+ Ve group, respectively (Fig. 1a). DOCA-salt treated adult offspring of LPS-treated mothers showed a significantly elevated systolic blood pressure (SBP) after 1 week of DOCA-salt treatment (LPS+ DOCA vs LPS+ Ve, P < 0.05) and the SBP continued to rise during the whole period of DOCA-salt treatment. The control offspring treated with DOCA-salt, however, just showed a slight increase of SBP after the first week of DOCA-salt treatment and showed statistical significance at the end of DOCA-salt treatment, as compared to that in control offspring with vehicle treatment (Con+ DOCA vs Con+ Ve, P < 0.05) (Fig. 1b, left panel). Remarkably, at the end of 4 weeks of DOCA-salt treatment, the increment of SBP in adult offspring of LPS-treated mothers was greater than that in control offspring (Fig. 1b, right panel). These results suggested a greater hypertensive response in offspring of prenatal exposure to inflammatory stimulus when challenged with second hypertensive risk factors.
We next determined the vascular structures of both thoracic aortas and superior mesenteric arteries by hematoxylin-eosin (HE) staining at the end of 4 weeks of DOCA-salt treatment. Data showed that compared with vehicle control, obvious increases in arterial vascular wall thickness, cross sectional area, wall:lumen ratio and asperous endothelial layers existed in thoracic aortas of adult offspring of LPS-treated mothers after DOCA-salt treatment (LPS+ DOCA vs LPS+ Ve, P < 0.05). Interestingly, we also noticed some slight changes in control offspring after DOCA treatment (Fig. 1c). In addition, the arterial wall thickness and cross sectional area significantly increased in adult offspring of LPS-treated mothers after DOCA treatment, as compared to these in DOCA-salt treated control offspring (LPS+ DOCA vs Con+ DOCA, P < 0.05). We also found similar changes in superior mesenteric arteries after DOCA-salt treatment ( Supplementary Fig. S1). Endothelium dysfunction, classically characterized by a decrease in endothelial nitric oxide synthase (eNOS) bioactivity and a lack of nitric oxide (NO) 22 , is associated with hypertension. As such, the expression of phosphorylated (p)-eNOS Ser1177 protein in thoracic aortas was determined by immunofluorescence staining at the end of 4 weeks of DOCA-salt treatment. The p-eNOS Ser1177 protein level in control offspring was not changed after DOCA-salt treatment, but decreased after DOCA-salt treatment in adult offspring of LPS-treated mothers (Fig. 1d). These results demonstrated that maternal inflammation results in higher vulnerability to vascular damage and hypertension in adult when suffered from hypertensive risk factors. Pregnant SD rats were administered intraperitoneally (i.p) with saline or lipopolysaccharide (LPS, 0.79 mg/kg) at gestational day (GD) 8, 10 and 12. Offspring were challenged with deoxycorticosterone acetate and high-salt diet (DOCA-salt) every other day (5-6 pm) for 4 weeks that started at the age of 16 weeks. (b) SBP was measured by a noninvasive tail-cuff method at 9-11 am the next day after DOCA treatment at indicated time points after DOCA-salt treatment (left panel). 0 wk means the day before the first time DOCA treatment. The increment of SBP at the end of 4 weeks DOCA-salt treatment in each group (∆ SBP) was shown in the right panel. n = 10 to 12 per group. (c) Hematoxylin-eosin (HE) staining of thoracic aortas and representative pictures from each group were shown. Vessel wall, nearby the arrow direction, represents endothelium. The values of vascular wall thickness, cross sectional area and wall:lumen ratio were quantified using NIS-Elements BR software Scientific RepoRts | 6:32642 | DOI: 10.1038/srep32642 Oxidative stress enhanced hypertensive response to DOCA-salt treatment in offspring of maternal inflammation via impairing the up-regulation of antioxidant capacity. Experimental models and circulating markers screening of systemic oxidative stress in human have suggested that oxidative stress promotes endothelial dysfunction, vascular remodeling, which correlates positively with SBP elevation 23 . Previous studies have shown that oxidative stress was implicated in traditional DOCA-salt induced hypertension model that with uninephrectomy 24 and also in adult offspring that had maternal exposure to nicotine and high-fat diet 25,26 . This raises the possibility that prenatal exposure to LPS impairs offspring's redox homeostasis and predisposes to DOCA-salt challenge. As such, we first determined the in situ O 2− level by dihydroethidium (DHE) staining of thoracic aortas. The fluorescence intensity was similar between vehicle-treated control and adult offspring of LPS-treated mothers and was not increased in control offspring after DOCA-salt treatment. However, the level of O 2− in adult offspring of LPS-treated mothers was higher than that in control offspring after 4 weeks of DOCA-salt treatment (LPS+ DOCA vs Con+ DOCA, P < 0.01) (Fig. 2a). As such, this finding supported the hypothesis that exaggerated oxidative stress exists in adult offspring of LPS-treated mothers after DOCA-salt treatment.
We further explored the potential mechanisms of exaggerated oxidative stress in thoracic aortas of offspring that received prenatal exposure to LPS after DOCA-salt treatment from two aspects: reactive oxygen species (ROS) generation and cellular antioxidant defense system, whose imbalance contributes to vascular oxidative stress 27 . Firstly, we measured the mRNA levels of NADPH oxidase subunits, the major enzyme of ROS generation in the artery wall, many of which are involved in hypertension 28 . The mRNA expression of these genes did not significant change, although some of them, such as Nox1, p22-phox, p47phox and p67phox (Ncf2) showed an increased trend in either control offspring or adult offspring of LPS-treated mothers after DOCA-salt treatment ( Supplementary Fig. S2a). These findings prompted us to investigate whether the anti-ROS capacity changes or not after DOCA-salt treatment by determining the expression of superoxide dismutases (SODs), heme oxygenase (HMOX-1), catalase, glutathione reductase-1(GPx-1), peroxiredoxins (Prdxs), and uncoupling protein 2 (UCP2), all of which have been involved in development of hypertension 29 . There were no significant changes of Gpx1, Hmox1, as well as Prdx3 mRNA expression in offspring of both control and prenatal exposure to LPS after DOCA-salt treatment, respectively ( Supplementary Fig. S2b). Surprisingly, the mRNA levels of Sod1 and Sod3 significantly decreased in thoracic aortas of adult offspring of LPS-treated mothers after DOCA-salt treatment, as compared to that in vehicle treated alone (LPS+ DOCA vs LPS+ Ve, P < 0.05). However, there were no significant changes of these two genes in thoracic aortas between DOCA-salt and vehicle treated control offspring (Con+ DOCA vs Con+ Ve, P > 0.05). Interestingly, the mRNA levels of Ucp2 and Prdx5 significantly increased in thoracic aortas in control offspring after DOCA-salt treatment (Con+ DOCA vs Con+ Ve, P < 0.05) but with no significant changes after DOCA-salt treatment in adult offspring of LPS-treated mothers (LPS+ DOCA vs LPS+ Ve, P > 0.05) (Fig. 2b). These data suggested an impaired ROS defense capacity in response to a second risk factor challenge in offspring of prenatal exposure to inflammatory stimulation.
To further confirm the role of oxidative stress in mediating hypertensive response in adult offspring of LPS-treated mothers when challenged with DOCA-salt, a thiol-containing antioxidant, N-Acetyl-L-cysteine (NAC) 30 was given simultaneously with DOCA-salt treatment. As expected, NAC administration significantly repressed the SBP elevation induced by DOCA-salt treatment in adult offspring of LPS-treated mothers (Fig. 3a). Consistent with these findings, antioxidant NAC prevented DOCA-salt induced increased artery wall thickness, cross sectional area as well as wall:lumen ratio and the damage to endothelial cell layers in adult offspring of LPS-treated mothers ( Fig. 3b and Supplemental Fig. S3a) and also rescued the p-eNOS Ser1177 protein level ( Fig. 3c and Supplementary Fig. S3b). Taken together, these results demonstrated that augmented oxidative stress resulting from the decreased antioxidant capacity is responsible for higher hypertensive response induced by DOCA-salt treatment in offspring of prenatal exposure to inflammatory stimulation and elimination of excessive ROS by NAC could be potent avenue to prevent vascular damage and the development of hypertension in PPH models.
Impaired PGC-1α recovery by continued elevation of NF-κB activity decreased ROS defense system in adult offspring of LPS-treated mothers in response to DOCA-salt challenge. There are several transcriptional factors that manipulate the balance between ROS and anti-ROS systems 15 . Among these genes, PGC-1α is a broad and powerful regulator of ROS metabolism through co-regulating the induction of proteins that participate in the cellular response to oxidative stress. For example, UCP2 has been proposed to be the direct downstream target of PGC-1α 31 and Prdx5 expression is mediated by PGC-1α direct association with its promoter regions 32 . Interestingly, knockdown of PGC-1α also significantly decreases SOD1 expression in response to oxidative stress 33 . In our previous data, DOCA-salt treatment led to abnormal expression model of UCP2, prdx5 and SOD1 in adult offspring of LPS-treated mothers (Fig. 2b), suggesting the critical role of PGC-1α in our model. To further address the role of PGC-1α in mediating enhanced oxidative stress in adult offspring of LPS-treated mothers after DOCA-salt treatment, we firstly determined the protein expression of (Nikon Corporation, Tokyo, Japan) and shown in the right panel. n = 6 per group. (d) Offspring were treated as described in (a) and the p-eNOS Ser1177 and eNOS protein levels were determined by immunofluorescence staining. Representative pictures selected from each group were shown. Vessel wall, nearby the arrow direction, represents endothelium. The relative fluorescence in each group was quantified using Image J software (right panel). n = 6 per group. Error bar represents S.E.M. *P < 0.05 or **P < 0.01, LPS+ DOCA vs LPS+ Ve at the same time point, respectively; # P < 0.05, LPS+ DOCA vs Con+ DOCA at the same time point; & P < 0.05, Con+ DOCA vs Con+ Ve at the same time point. Two-way ANOVA.
Scientific RepoRts | 6:32642 | DOI: 10.1038/srep32642 PGC-1α at the end of DOCA-salt treatment. As expected, the protein level of PGC-1α significantly increased after DOCA-salt treatment in thoracic aortas of control offspring (Con+ DOCA vs Con+ Ve, P < 0.05), which was similar with PGC-1α expression response to H 2 O 2 stimulation challenge as a compensatory mechanism for anti-oxidative stress 33 . However, there was no difference in the expression level of PGC-1α between vehicle and DOCA-salt treated adult offspring of LPS-treated mothers (LPS+ DOCA vs LPS+ Ve, P > 0.05). Besides, the basal level of PGC-1α protein in thoracic aortas of adult offspring of LPS-treated mothers was significantly higher than that in control offspring (LPS+ Ve vs Con+ Ve, P < 0.05) (Fig. 4a). The consistent expression pattern of UCP2, Prdx5 (Fig. 2b) and PGC-1α ( Fig. 4a) in our model, strongly suggested that the compensatory action of PGC-1α -mediated enhanced ROS defense system occurs in adult offspring of prenatal exposure to inflammatory stimulation at rest state, but loses its further compensatory capability after a second oxidative stressor challenge. However, it raised an interesting question as to why no PGC-1α increment was observed in offspring of prenatal exposure to inflammatory stimulus after exposure to a hypertensive risk factor?
Several factors affect PGC-1α expression under oxidative stress or inflammatory challenge, such as mitogen activated protein kinase (MAPK) and NF-κ B 34 . Among these, MAPK signal pathway has been involved in response to various environmental stresses 35 . Since a previous study found that MAPK signal pathway was activated in traditional DOCA-salt hypertensive rats 36 , we first determined the MAPK signaling in the thoracic aortas of adult offspring of LPS-treated mothers after DOCA-salt treatment. We found that the expression levels of p-p38 MAPK, p-ERK1/2 and p-JNK were not changed in thoracic aortas of both control offspring and adult offspring of LPS-treated mothers after 4 weeks DOCA-salt treatments ( Supplementary Fig. S4). Besides, the critical transcriptional factor of inflammatory response, NF-κ B, suppresses PGC-1α expression after activated by inflammatory cytokines 37 . Since we previously found increased inflammatory cytokines and NF-κ B activation in both kidney and thoracic aortas of adult offspring of LPS-treated mothers 7,10 , we assessed the activity of NF-κ B by measuring the protein levels of phosphorylated (p)-p65 Ser536 . Interestingly, the p-p65 Ser536 level peaked in thoracic aortas of adult offspring of LPS-treated mothers after 4 weeks of DOCA-salt treatment, compared to either DOCA-salt treated control offspring or vehicle treated adult offspring of LPS-treated mothers (LPS+ DOCA vs Con+ DOCA and LPS+ DOCA vs LPS+ Ve, P < 0.05, Fig. 4b). In support of this finding, the expression levels of genes that are (a) NAC was given simultaneously with DOCA-salt for 4 weeks in both control (Con+ DOCA+ NAC group) and adult offspring of LPS-treated mothers (LPS+ DOCA+ NAC group) and SBP was determined by a noninvasive tail-cuff method. (b) Offspring were treated as described in (a) and HE staining of thoracic aortas and representative pictures from each group were shown. Vessel wall, nearby the arrow direction, represents endothelium. The values of vascular wall thickness, cross sectional area and wall:lumen ratio were quantified using NIS-Elements BR software and were shown in Supplemental Fig. S3a. (c) Offspring were treated as described in (a) and the p-eNOS Ser1177 and eNOS protein levels were determined by immunofluorescence staining. Representative pictures selected from each group were shown. Vessel wall, nearby the arrow direction, represents endothelium. The value of fluorescence was quantified using Image J software and shown in supplemental Fig. S3b. Error bar represents S.E.M. *P < 0.05 and **P < 0.01 denote the statistical comparison between the two marked treatment groups, respectively. n = 6-7 per group for (a-c). Two-way ANOVA. Representative plots in each group and statistical data of relative densitometry, normalized by β -actin, were shown. n = 6 per group for (a-c). Error bar represents S.E.M. *P < 0.05 and **P < 0.01 denotes the statistical comparison between the two marked treatment groups, respectively. Two-way ANOVA.
A previous study reported that renal PGC-1α expression was suppressed at the beginning of LPS-induced sepsis and reversed to the normal level during the renal functional recovery and the pattern of PGC-1α expression was believed to be necessary for renal functional recovery after sepsis 38 . This prompted us to determine whether the PGC-1α expression followed the similar pattern at different time-points after DOCA-salt treatment in offspring of both control and adult offspring of LPS-treated mothers and whether NF-κ B over-activation was involved in inhibiting PGC-1α expression. In control offspring, we found that the PGC-1α protein expression decreased after 3 days of DOCA-salt treatment but soon rebounded to the initial level by 7 days of DOCA-salt treatment, while p-p65 Ser536 showed the opposite pattern (Fig. 4c, left panel). However, in adult offspring of LPS-treated mothers, the PGC-1α expression didn't rebound to the initial level by 7 days of DOCA-salt treatment, meanwhile, p-p65 Ser536 levels continued to increase from 3 days to 7 days after DOCA-salt treatment (Fig. 4c, right  panel). All these data indicated that prenatal exposure to LPS predisposes adult offspring to NF-κ B activation and further augmented activation lead to retarded recovery of PGC-1α expression during stressor challenge.
Inhibition of NF-κB activity by TPCK restores rapid PGC-1α recovery and prevents SBP elevation in adult offspring of LPS-treated mothers in response to DOCA-salt challenge. To further confirm our above hypothesis that the over-activated NF-κ B signal pathway in repressing PGC-1α expression in adult offspring of LPS-treated mothers after DOCA-salt treatment, a specific NF-κ B inhibitor, N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) 39 , was given simultaneously with DOCA-salt treatment. As expected, TPCK prevented the increased p-p65 Ser536 level in thoracic aortas of both control offspring and adult offspring of LPS-treated mothers by 3 or 7 days of DOCA-salt treatment. Importantly, TPCK restored PGC-1α expression at 3 days or 7 days after the initial DOCA-salt treatment in thoracic aortas of offspring that underwent prenatal exposure to LPS, respectively (Fig. 5a). TPCK also restored UCP2, SOD1 and SOD3 expression 7 days after the initial DOCA-salt treatment in thoracic aortas of adult offspring of LPS-treated mothers (Fig. 5b). In agreement with the above findings, TPCK administration significantly repressed the SBP increment induced by 7 days of DOCA-salt treatment in adult offspring of LPS-treated mothers (Fig. 5c). All this data demonstrated that prenatal inflammation exposure leads to the proneness of NF-κ B activation at resting state and continued elevation of NF-κ B activity results in suppressed PGC-1α rebound when facing a stressor challenge, which might be a pivotal factor implicated in vascular damage and progressive development of hypertension via an excessive oxidative stress mechanism.

Discussion
The early onset of CVD is dramatically increasing in the past several years. More than 17% of deaths caused by CVD occurred before the age of 60 in 2012 40 . As a major cardiovascular risk factor, the incidence of raised blood pressure (defined as systolic and/or diastolic blood pressure ≥ 140/90 mmHg) in adults aged 18 years and over is around 22% 40 . However, hypertension development begins decades before the onset of clinical manifestations and may be hastened by maternal detrimental factors 20,41 . Therefore, understanding the mechanisms of hypertension that result from fetal programming will provide new interventional strategies for reducing the prevalence of hypertension. The current study has analyzed the susceptibility of hypertension in response to DOCA challenge in offspring of prenatal exposure to inflammation and the major findings are as follows: (1) prenatal exposure to LPS resulted in higher blood pressure and more severe vascular damage in adult offspring after postnatal 4 weeks of DOCA-salt treatment; (2) inadequate anti-oxidant defense capacity caused by impaired PGC-1α rapid recovery was implicated in vascular damage and blood pressure elevation in response to DOCA-salt treatment via oxidative stress in adult offspring of LPS-treated mothers; (3) mechanically, prenatal inflammation exposure compensatory increase in PGC-1α expression which in turn up-regulated the antioxidant capacity and maintained the O 2− below the subliminal level that could not cause obvious vascular damage in offspring's thoracic aortas at a resting state. However, DOCA-salt treatment induced a sustained elevation of NF-κ B activity and impaired the rapid PGC-1α recovery in thoracic aortas of offspring that received prenatal exposure to LPS, which may have a pivotal role in the early onset of hypertension.
Growing evidence has shown that exposure to detrimental factors during pregnancy sensitizes offspring to damage in adult 3 . For examples, viral infection during pregnancy increases the frequency of autism spectrum disorder in offspring 42 , maternal hyper-cholesterolaemia during pregnancy promotes offspring to develop atherosclerotic lesions at earlier age 43 and high-fat diet feeding during pregnancy and lactation augmented offspring's lung inflammation and remodeling 44 . Previous studies showed that 0.79 mg/kg LPS administration at gestational day 8, 10 and 12 caused a very low percentage of fetal anomalies with no obvious changes in abortion rates 45,46 . We previously found that this dose of LPS injection led to a slight blood pressure elevation but showed no significant effect on abortion, the size of pups and/or sex ratio of the litter 8,47 . The current study focuses on the effects of maternal inflammation on adult hypertension development. We found that higher blood pressure and more severe vascular damage in response to DOCA-salt stimulation in adult offspring of LPS-treated mothers, which adds more evidence for the concept of in utero origins of hypertension 48 . The traditional model of DOCA-salt induced hypertension in which the blood pressure increment is mainly mediated by increased intravascular volume and circulating catecholamines is dependent on unilateral nephrectomy 49 . Though renal function plays a critical role in maintaining normal blood pressure, the process of clinical hypertension development in respect to renal dysfunction could not be well imitated by unilateral nephrectomy in animal study 50 . The adult offspring of LPS-treated mothers in the current study showed increased inflammatory status and oxidative stress, although the blood pressure increment did not reach the high level to that obtained in DOCA-salt treatment plus nephrectomy.  These offspring were prone to hypertension and vascular damage in the absence of nephrectomy when challenged with DOCA-salt. Epidemiological studies of primary hyper-aldosteronism being an important risk factor for hypertension 18,19 encourage us to believe that DOCA-salt treatment without nephrectomy by using the adult offspring of LPS-treated mothers might be a good animal model for studying the mechanisms of hypertension development and also for pharmacodynamics evaluation of anti-hypertension drugs.
The progression of cardiovascular disease is usually accompanied by imbalance between generation and elimination of ROS, such as hypertension and atherosclerosis 51 . Previous studies showed that traditional DOCA-salt rats showed increased aortic O 2− production 52 via increased expression of NADPH oxidase subunits p22phox, together with decreased anti-oxidant genes expression 24 . In the current study, despite the increased trend in the expression of NADPH oxidase subunits in control offspring after DOCA-salt challenge, the O 2− level was not significantly increased. This might be attributed to a healthy self-compensatory machinery of upregulated anti-oxidant defense capacity, as evidenced by the increased expression of UCP2 and Prdx5, though without any changes of SODs expression. However, there were not any induction of UCP2 and Prdx5 transcription, even with a significant reduction of SOD1 and SOD3 transcription, which is critical for the clearance of excessive ROS 53 , after DOCA-salt challenge in the thoracic aortas of offspring that received prenatal exposure to inflammation. The antioxidant NAC, which is an excellent source of sulfhydryl groups acting as a potential free radical scavenger 30 , could alleviate DOCA-salt induced blood pressure elevation and vascular damage in adult offspring of LPS-treated mothers. These results demonstrate that impaired motivation of anti-oxidant system existed in offspring of prenatal exposure to inflammation when suffering from secondary risk factors. A previous study showed that maternal high-fat diet repressed the expression of anti-oxidant defense genes in liver of the male offspring rats 26 . Furthermore, modification in antioxidant activity by maternal low protein diet could predispose to pancreatic islet dysfunction 54 . Our findings together with previous studied strongly suggested that the potential programming of ROS and anti-ROS system prenatally may be a vital risk factor for adult health in offspring of prenatal exposure to detrimental factors 26,54 .
PGC-1α is a crucial regulator in response to various stressors and associates with the development of several diseases 55 . Epidemiological studies have revealed that a common Gly482Ser polymorphism of PGC-1α is associated with arterial hypertension 56 . In addition, PGC-1α has also been reported to prevent the migration of vascular endothelial cells and smooth muscle cells and prevent endothelial dysfunction 57,58 . This may have an important role for PGC-1α in hypertension development. In our current study of control offspring, the protein level of PGC-1α was significantly increased after 4 weeks of DOCA treatment, which correlated with the increased expression of its direct target genes UCP2 and Prdx5 31,32 , supporting the critical role of PGC-1α in response to stressors. However, prenatal inflammatory stimulation led to a pre-existing higher level of PGC-1α in adult offspring at resting state, without any additional elevation after 4 weeks of DOCA treatment. The pre-existing higher level of PGC-1α might be an adaptive response to inflammation response 17 during the prenatal stage and become programed in adult life, which in turn raises the threshold of anti-ROS response when suffering from a persistent weak ROS inducer.
Previous reports have revealed that a rapid PGC-1α rebound plays a protective role in renal functional recovery after sepsis 38 . Our current study also observed a similar trend in control offspring after DOCA-salt treatment. However, this rapid PGC-1α rebound did not exist in offspring of prenatal exposure to inflammation after DOCA-salt challenge. This demonstrates that the impaired rapid PGC-1α rebound, induced by prenatal exposure to inflammation or other detrimental factors, which was accompanied by increased ROS accumulation, renders the offspring more sensitive to vascular damage and the development of hypertension.
Mechanistically, we found that continued NF-κ B over-activation contributed to the repressed PGC-1α rapid rebound in responding to DOCA-salt challenge in offspring of prenatal exposure to inflammation. This was consistent with previous studies that PGC-1α mRNA levels was markedly reduced after LPS injection and inflammation-dependent PGC-1α repression was rescued by inhibiting the NF-κ B pathway 16,59 . This rapid rebound of PGC-1α expression in parallel with rapid NF-κ B recovery at the early stage of DOCA-treatment in control offspring with DOCA-treatment suggests that they may antagonize each other, consistent with the slightly increased blood pressure and vascular damage. However, this mutual inhibition of them at the early stage of DOCA-salt challenge was substituted with persistent up-regulation of NF-κ B activation during the whole period of DOCA-salt treatment in thoracic aortas of offspring that received prenatal exposure to inflammation, accompanied by retarded PGC-1α rebound. This NF-κ B over-activation might attribute to the impaired NF-κ B self-negative feedback loop on newly Iκ Bα re-synthesis in the thoracic aortas of offspring that received prenatal inflammatory exposure 13 . This over-activation of NF-κ B on inhibiting PGC-1α expression was further confirmed following the application of a NF-κ B inhibitor, TPCK, which reversed the PGC-1α rebound, up-regulated the expression of anti-oxidant genes UCP2 and SOD1, as well as prevented blood pressure elevation after 7 days of DOCA-salt treatment. Therefore, proneness to NF-κ B over-activation, programmed by prenatal inflammatory stimulation, plays a critical role in interrupting PGC-1α -mediated antioxidant capacity when responding to detrimental factors in adults.
Collectively, the present study has established a new re-stimulation model for studying the potential mechanisms of the early onset of hypertension by using offspring that received prenatal inflammatory exposure and postnatal DOCA-salt re-stimulation. Prenatal programmed proneness to persistent NF-κ B over-activation impairs the role of PGC-1α on up-regulating antioxidant capacity, thereby having a critical role in the early onset of hypertension when responding to a second hypertensive risk factor. Since prenatal exposure to inflammation is an important unresolved public health problem worldwide and easily acquired experience of cardiovascular risk factors in adulthood, the present findings provide a new notion by which prenatal inflammatory stimulus contributes to the increased incidence of adult hypertension. New strategies based on restraining programmed NF-κ B over-activation or antioxidant capacity will provide new targets for preventing the worldwide epidemic of hypertension. Prenatal inflammatory stimulation model. Time-dated pregnant SD rats (250 g to 300 g) were randomly divided into two groups as described previously 8 . The pregnant rats were intraperitoneally administered with saline (Control group) or LPS (0.79 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) (LPS group), respectively, on gestational day (GD) 8, 10 and 12.
Postnatal DOCA-salt stimulation model. Male pups from the aforementioned control and LPS group were subjected to 1% NaCl in the drinking water with subcutaneous injection of DOCA (20 mg/kg, Wako Pure Chemical Industries Ltd, Osaka, Japan) every other day from post-birth week 16 to week 20, defined as Con+ DOCA and LPS+ DOCA group, respectively. Control and LPS group treated with 50% glycerol subcutaneously were taken as vehicle control, defined as Con+ Ve and LPS+ Ve group, respectively. For NAC (Sigma-Aldrich) treatment, NAC (0.5 g/L, estimated oral intake of 187 mg/kg/d) in the drinking water was given to Con+ DOCA and LPS+ DOCA group for 4 weeks, defined as Con+ DOCA+ NAC and LPS+ DOCA+ NAC group, respectively. For TPCK (Sigma-Aldrich) 39 treatment, TPCK (3 mg/kg,) was given daily to Con+ DOCA or LPS+ DOCA group intraperitoneally, defined as Con+ DOCA+ TPCK and LPS+ DOCA+ TPCK group, respectively. SBP measurement. SBP was measured by the noninvasive tail-cuff method with computer-assisted BP-2010 Series tail measurement equipment (Softron Beijing Biotechnology Co., Ltd, Beijing, China). The operation procedure was followed as described previously 8 . Briefly, rats were placed inside a warming chamber (~34 °C) for 15 minutes before measurement of artery blood pressure and then were placed in plastic restraints. A cuff with a pneumatic pulse sensor was attached to the tail. SBP was calculated from three consecutive recordings. The measurement of blood pressure was performed 9 to 11 am at the next day after DOCA treatment at indicated time points. 0 week means the day before the first time of DOCA treatment. The investigators were blinded for measuring the blood pressure.
Histological analysis. Histological structures of thoracic aortas and superior mesenteric arteries were determined by standard HE staining. The coverslips were visualized under a Leica confocal laser-scanning microscope (Leica, Wetzlar, Germany). The values of vascular wall thickness, cross sectional area and wall:lumen ratio were quantified using NIS-Elements BR software (Nikon Corporation, Tokyo, Japan), as described elsewhere 60 . The investigators were blinded for acquiring the images.
Immunofluorescence staining. The protein levels of p-eNOS Ser1177 and eNOS in thoracic aortas were determined by immunofluorescence staining as described previously 10 . Donkey Anti-Rabbit IgG H&L (Alexa Fluor ® 647) (Abcam, Cambridge, UK) was used for an additional 1 hour incubation after anti-p-eNOS Ser1177 or anti-eNOS (Abcam, Cambridge, UK) incubation and washing. Sections were then incubated with 1 mg/ml DAPI for 10 min, washed three times with PBS, and mounted into Vectashield ® mounting medium (Vector Laboratories, Burlingame, CA). The coverslips were visualized under a Leica confocal laser scanning microscope (Leica, Wetzlar, Germany). The investigators were blinded for acquiring the images. The value of fluorescence was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

Real-time RT-PCR.
Real-time RT-PCR was performed as described previously 61 . Total RNA was extracted from thoracic aortas using Trizol (Roche Molecular Biochemicals, Mannheim, Germany) and total RNA (1 μ g) was then reverse-transcribed into cDNA using a First Stand cDNA Synthesis Kit (DBI Bioscience, Ludwigshafen, Germany). Each Real-time PCR reaction was carried out in a total volume of 20 μ l with Quanti Tect SYBR Green PCR Master Mix (MJ Research) according to the following conditions: 2 min at 95 °C, 40 cycles at 95 °C for 10 s, 60 °C for 15 s, 68 °C for 10 s, 72 °C for 20 s, using the ABI Prism 7700 sequence detection system (ABI). The cycle threshold (Ct) values were normalized by the internal control β -actin. Primer sequences for real-time RT-PCR, obtained from reported literatures or designed by Pubmed Primer-BLAST, were listed in Supplementary Table S1.
In Situ O 2− detection. O 2− level was measured by DHE (Invitrogen, Carlsbad, CA, USA) staining, according to a previous report with some modification 62 . Briefly, cross segments of thoracic aortas were placed in a phosphate buffer solution buffer and allowed to equilibrate for 30 minutes at 37 °C, followed by incubated with 5 μ mol/L DHE for additional 20 minutes at 37 °C in dark. After incubation, segments were washed with warmed phosphate buffer solution for 5 minutes X 3 times at 37 °C and then quickly imaged under a Leica DM4000B microscope (Leica). The value of DHE fluorescence was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
Statistical analysis. Data are expressed as means ± S.E.M. Significance of difference in mean values was determined using a two-way ANOVA analysis of variance followed by the Bonferroni post hoc test. A P value less than 0.05 was considered to be significant.