Mechanism of Earthquake Simulation as a Prenatal Stressor Retarding Rat Offspring Development and Chinese Medicine Correcting the Retardation: Hormones and Gene-Expression Alteration

We aimed to investigate the mechanism of shaking as a prenatal stressor impacting the development of the offspring and Chinese medicines correcting the alterations. Pregnant rats were randomized into earthquake simulation group (ESG), herbal group (HG) which received herbal supplements in feed after shaking, and control group (CG). Findings revealed body weight and open field test (OFT) score of ESG offspring were statistically inferior to the CG and HG offspring. The corticosterone levels of ESG were higher than those of CG but not than HG. The dopamine level of ESG was slightly lower than that of the CG and of HG was higher than that of ESG. The 5-HT of ESG was higher than CG and HG. The growth hormone level of the ESG was significantly lower than ESG but not than CG. Gene expression profile showed 81 genes upregulated and 39 genes downregulated in ESG versus CG, and 60 genes upregulated and 28 genes downregulated in ESG versus HG. Eighty-four genes were found differentially expressed in ESG versus CG comparison and were normalized in ESG versus HG. We conclude that maternal shaking negatively affected physical and nervous system development, with specific alterations in neurohormones and gene expression. Chinese herbal medicine reduced these negative outcomes.


Introduction
Maternal effects have been demonstrated as an essential factor for offspring development in many species. Because of the long period of perinatal mother-infant interaction in mammals, the growth and development and variations of offspring are very likely to be influenced by maternal impacts, leaving long-term consequences for both psychological and physiological health [1]. Recent human studies have shown that long-lasting and a wide variety of prenatal stressors, from anxiety and partner relationship problems to natural disasters, increase the risk for a diverse range of adverse neurodevelopmental outcomes in the child, including impaired cognitive development and behavioral problems [2,3]. Animal experiments have convincingly demonstrated that prenatal maternal stress affects pregnancy outcome and results in early programming of brain functions with permanent changes in neuroendocrine regulation, gene expression, and behavior in offspring [4]. Prenatal restraint stress in rats is a common experimental model of early stress known to have long-term behavioral and neurobiological consequences [5,6]. PS modifies the plastic responses of the adult brain, including the circuitry of the hippocampus-hypothalamuspituitary-adrenalaxis (HHPA), that participate in the neuroendocrine control of feeding and metabolism in adult life [7]. Evidence-Based Complementary and Alternative Medicine As a typical prenatal stress, shaking can significantly impact the psychological and intellectual development of fetus and birth outcomes [8] in human. Naturally, earthquake is a fierce shaking. Tan et al. [9] reported that rates of birth defects after an earthquake were significantly higher than those before earthquake, whose spectrum was dramatically altered after earthquake, with the markedly increased occurrences of ear malformations; meanwhile the ratio of preterm birth after earthquake was significant increased than that of before earthquake. Oyarzo et al. [10] reported that women exposed to the February 27th 2010 Chilean earthquake during her first trimester delivered smaller newborns and they were more likely diagnosed with early preterm delivery, preterm delivery, and PROM but were less likely diagnosed with intrauterine growth retardation and late delivery compared to those exposed at third trimester, indicating disasters such as earthquakes are associated to adverse perinatal outcomes that impact negatively the entire maternal-neonatal healthcare system. Like the other alterations induced by PS in behavior those in learning and their direction appears to be dependent on the intensity, duration, and timing of the maternal stress [11].
In Chinese medicine, PS from shaking or an analog of earthquake is considered as a factor which impairs kidney Qi (shen qi) [12]. As kidney is the root of earlier heaven (the congenital constitution), it governs reproduction and development and holds oriffice of labor, whence agility and emanates. Jin Kui Shen Qi Wan (JKSQW) is a typical herbal formula supplementing kidney Qi, which recovers the physiological functions of kidney [13].
The current study involves shaking as a prenatal stressor. A first goal was to establish that earthquake simulation led to significant delays in development. A second goal was to examine whether Chinese traditional medicine could be used to address these negative effects. Based on the above information, we hypothesized parental kidney is injured from PS derived from earthquake simulation on rats, traits are handed down to offspring, showing development retardation; JKSQW could recover the dysfunctions of kidney whose underlying mechanism could involve development, hormones and gene expression alterations.

Grouping.
Forty-five Sprague-Dawley (SD) female rats (230 g ∼270 g) and 45 male rats (225 g ∼261 g) were involved in this research. The rats were housed in a room with a temperature of 22 • C, 12 hour light/dark cycle and fed with food and water ad libitum. After a week of adaption housing, the female rats were mated with the male rats. Pregnancy was confirmed by vaginal plug test. Then the 34 pregnant rats were randomized into three groups, control group (CG) (n = 11), earthquake simulation with conventional chow group (ESG) (n = 11), and earthquake plus herbal group (HG) (n = 12), and they were housed under pregnant rat cages until the delivery. With this procedure, all the groups were transferred with equivalent stress during pregnancy. There was no statistical difference of gestation time detected or body weight of the first day of gestation (CG: 234.87±2.20, ESG: 234.98 ± 1.95, and HG: 235.16 ± 1.96, ANOVA test, P > 0.05 (g)) in the three groups. After delivery, all the litters of the three groups were housed with their mothers until the 25th day after birth.

Earthquake
Simulation. The ESG cages housing pregnant rats were manually shaken up and down 3 times to simulate an initial earthquake and then were shaken for 50 timesover the next 15 minutes to modulate an aftershock [14]. The shaking was performed twice a day until delivery. Severity of the shake was measured with a seism velometer (DX-6Y2, Cheng Du Mei Huan Tech. Co. Ltd.), showing 9.6∼10.5 of seismic intensity, 950 mg ∼1050 mg of vertical peak ground accelerations (PGA), which was similar to the PGA (1080 mg) of Wenchuan earthquake, May 12, 2008, China. , and Cortex Cinnamomi Cassiae (Rou Gui)) bought from Tong Ren Tang Technologies, Co., Ltd. The pill of JKSQW was grinded and added to the conventional feed 0.5∼0.6 g/d.

Open Field Test (OFT).
A square board (90 cm × 90 cm) painted with yellow and white squares (15 cm × 15 cm). The offspring of 25 days old was placed in the center of the board. We counted how many squares the offspring had crawled across in two minutes. One score was given only when the four paws of an offspring were in one square.

Gene Expression
A Mann-Whitney test showed significant difference between the three groups on the OFT (Mann-Whitney U = 1448.500, Wilcoxon W = 2529.500, Z = −3.819, P = 0.000) ( Figure 2): the OFT scores of HG and CG were both significantly higher than those observed in the ESG.

Hormone Levels.
The corticosterone levels of CG was statistically lower than ESG and slightly than HG (Figure 3(a)). The dopamine level of ESG was slightly lower than the CG and of HG was significantly higher than the ESG (Figure 3(b)). The 5-HT of ESG showed a highest level and the CG lowest (Figure 3(c)). The growth hormone level of the HG was statistically higher than the CG and ESG (Figure 3(d)).  Figure 4), among which 14 GO annotations were obtained including, ligase activity, regulation of metabolic process, positive regulation of metabolic process, cellular component assembly, membrane bounded organelle, biosynthetic process, cellular component biogenesis, and cellular response to stimulus. (Table 4 (Supporting Information 2)), and among which 12 KEGG pathways were annotated, including oocyte meiosis, vascular smooth muscle contraction, RIG-I-like receptor signaling pathway, longterm potentiation, ubiquitin mediated proteolysis, and longterm depression (Table 5).

ESG versus HG.
Gene expression profile showed 60 genes upregulated and 28 genes downregulated (P < 0.01) in ESG versus CG (Table 6 (Supporting Information 3), Figure 5), among which five GO annotations were obtained including protein complex localization, cellular component assembly, cellular component biogenesis, anatomical structure formation, and organelle lumen (Table 7), and among which 5 KEGG pathways were annotated, including cell cycle, Jak-STAT signaling pathway, Type II diabetes mellitus, One carbon pool by folate, and insulin signaling pathway (Table 8).
No genes were found, which were significantly differently expressed simultaneously in ESG versus CG and ESG versus HG. However, 8,426 genes were found no statistical difference in HG versus CG (P > 0.05) among which 84 were found also presented in the differently expressed genes in ESG versus HG (Table 9 (Supporting Information 4)).    KEGG annotation. As showed in Figure 6(a), Irf7, Ninj2, and Isca1 were significantly hypoexpressed in ESG (FC < 0.5); however, the gene expression of Plxnc1 did not match the RT-PCR validation; in Figure 6(b), the four genes were not significantly hypoexpressed in HG versus CG (0.5 < FC < 2), and the RT-PCR validation showed an obviously reduced ΔΔCt values compared with those in Figure 6(a). The gene expression profile chip outcomes showed a favorable match with the RT-PCR result.

Discussion
Substantial evidence from preclinical laboratory studies indicates that PS affects the hormonal and behavioral development of offspring. PS has been found to alter baseline and stress-induced responsivity of the HPA axis and levels and distribution of regulatory neurotransmitters, such as norepinepherine, dopamine, serotonin, and acetylcholine and to modify key limbic structures and to retard intrauterine growth [15]. In this study, ESG demonstrated differences from CG on body weight, hormone levels, and gene expressions, and HG differed from the ESG group on body weight, hormone levels, and gene expressions. From the perspective of Chinese medicine, once parental kidney is injured from PS, manifestations are handed down to offspring, showing development retardation and OFT performance reduction. JKSQW is a typical herbal formula for kidney qi supplementing, which recovers the physiological functions of kidney. In this study, the body weight and OFT performance were improved by JKSQW, supporting the effectiveness of Chinese herb remedy in rodents in lab [13]. Experimentally, PS in animal models mal-programs offspring physiology, resulting in increasing the likelihood of disorders of HPA axis activity and anxiety-related behaviors in adulthood [16]. PS increases plasma levels of corticosterone and corticotrophin releasing hormone in the mother and fetus, which may contribute to insulin resistance and behavior disorders in their offspring that include attention and learning deficits, generalized anxiety and depression [17]. We demonstrated that the serum corticosterone of ESG were significantly higher than CG and slightly higher than HG, which was in accordance with previous reports [18][19][20]. Animal studies indicate that PS can affect the activity of the placental barrier enzyme 11-βHSD2 (11β-hydroxysteroid dehydrogenase type 2), which metabolizes corticosterone [2,17]. 5-HT level of ESG was significantly higher than CG and HG. Alterations in activity of the central 5-HT system play an essential role in many of these behavioral aberrations due to PS [21,22]. During pregnancy, the 5-HT system has a fundamental role in the fetus' development of the central nervous system, and 5-HT neurotransmission is involved in the activation and feedback of HPA axis throughout life [23]. Huang et al. [14] reported that levels of 5-HT were higher in rat hippocampus and hypothalamus of fetuses in the CUS group, that is, chronic unpredictable stress maternally performed than in the controls. Increased 5-HT signaling increases the expression of key transcription factors, notably nerve growth factor induced protein A, which binds to and regulates activation of the GR promoter [24]. No difference of the dopamine level between ESG and CG were obtained, indicating earthquake may not alter the offspring dopamine. Interestingly JKSQW in HG significantly elevated the dopamine level of ESG. Carboni et al. [25] reported prenatal catecholamine stimulation was obtained by amphetamine or nicotine. We observed that PS did not change dopamine. No difference of the hormone level between ESG and CG were obtained, indicating earthquake may not impact on the growth hormone of offspring. Interestingly, however, JKSQW in HG significantly elevated the dopamine level of ESG, which might be explained by the function of kidney that governs development. Shen and Cai [26] reported that growth hormone genes were downregulated in a kidney-qi deficiency rat model and Chinese formula supplementing kidney qi could correct the downregulation. Mak et al. [27] found that chronic kidney disease in children was     associated with dramatic changes in the growth hormone and insulin-like growth factor (IGF-1) axis, resulting in growth retardation. Yang and Li [28] reported that JKSQW could recover the downregulated growth hormone genes (Somatotropin precursor, NM-008117) in a kidney-yang deficiency rat model. Researches of the neurobiological mechanisms underlying the interaction between PS and adult mental disorders suggest the involvement of multiple neurotransmitter systems [29,30]. Findings of the hormones alterations suggest manual earthquake is a liable model modulating the fear from natural earthquake involving development retardation and neurotransmitter systems disorder. Meanwhile, from the perspective of Chinese medicine, kidney function is disturbed by the earthquake and recovered by JKSQW.
We found 81 genes upregulated and 39 genes downregulated in ESG versus CG, from which 14 significant GO and 12 KEGG pathways were annotated, indicating diversified and complicated physiological and psychological impacts on offspring left by the prenatal earthquake as a prenatal stress, for example, long-term depression and long-term potentiation. Mychasiuk et al. [31] reported that significant gene expression level changes in 558 different 14 Evidence-Based Complementary and Alternative Medicine   16 Evidence-Based Complementary and Alternative Medicine genes, associated with overrepresentation of 36 biological processes and 34 canonical pathways indicating prenatal stress did not have to be experienced by the mother herself to influence offspring brain development. Among the GO annotations Itpr1 and Itpr2 appeared in almost all the affected pathways. In nonexcitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca 2C channel, which plays a major role in Ca 2C signalling. Three isoforms of IP3R have been identified (IP3R-1, IP3R-2, and IP3R-3) and most cell types express different proportions of each isoform [31]. IP3Rs play major roles in agonistsinduced intracellular Ca 2C release and also in store operated Ca 2C entry, a process whereby the depletion of intracellular Ca 2C store causes the opening of Ca 2C channels in the plasma membrane [32]. The intracellular Ca 2+ elevations induced by BDNF required a signaling pathway consistent with the activation of the Trk-IP3R cascade, which was also necessary for the activation of the membrane conductance IBDNF [33,34]. Amaral and Pozzo-Miller [35] reported that Trk receptors, IP3Rs, full intracellular Ca 2+ stores and Ca 2+ influx are all required for BDNF-induced Ca 2+ elevations and membrane currents. Opposing influences of mBDNF and proBDNF on long-term potentiation and long-term depression might contribute to the dichotomy of BDNF actions on behaviors mediated by the brain stress and reward systems [36,37]. Twelve KEGG pathways were annotated, including oocyte meiosis, vascular smooth muscle contraction, RIG-I-like receptor signaling pathway, long-term potentiation, ubiquitin mediated proteolysis, and long-term depression, Titterness and Christie [38] prenatal ethanol and prenatal stress produce sex-specific alterations in synaptic plasticity in the adolescent hippocampus. Calpains, which belong to a family of at least 14 members of calcium-dependent cysteine proteases and are involved in apoptosis are implicated in a wide range of physiological functions including cell motility, differentiation, signal transduction, including cell survival pathways, cell cycle progression, regulation of gene expression, and long-term potentiation [39,40]. Yang et al. [41] reported that prenatal stress (10 unpredictable, 1 s, 0.8 mA foot shocks per day during gestational days [13][14][15][16][17][18][19] impaired long-term potentiation (LTP) but facilitated long-term depression (LTD) in hippocampal CA1 region in slices of the prenatal stressed offspring (5 weeks old). Proteolysis by the ubiquitin-proteasome pathway has attained prominence as a new molecular mechanism which regulates varied important functions of the nervous system, including development of synaptic connections and synaptic plasticity through control of axonal growth, axonal and dendritic pruning, and regulation of synaptic size and number [42].
We found 60 genes upregulated and 28 genes downregulated in HG versus ESG, from which five significant GO and five KEGG pathways were annotated, indicating diversified cellular biological process and signaling pathways. Interestingly, Socs 2 and Socs 4 of Socs (suppressors of cytokine signaling) family appeared in three of the KEGG pathways. SOCS family consists of eight structurally similar proteins (SOCS-1 to SOCS-7 and CIS), which have been implicated as potential inhibitors of tissue growth during both prenatal and postnatal life [43] and their actions clearly now extend to other intracellular pathways, they remain key negative regulators of cytokine and growth factor signaling [44]. Cytokinemediated JAK/STAT signaling, that is, Janus kinase/signal transducers and activators of transcription, controls a number of vital biologic responses, including immune function, cellular growth, differentiation, and hematopoiesis [45]. The SOCS Family-The SOCS proteins were identified as STAT target genes that directly antagonize STAT activation, resulting in a classic "feedback loop" [46]. PS in rats induced lifespan reduction of neurogenesis in the dentate gyrus and produced impairment in hippocampal-related spatial tasks through blocking the increase of learninginduced neurogenesis [47]. Previous research reported that male rats exposed to stress in utero are characterized by a decrease in hippocampal cell proliferation, and consequently neurogenesis, from adolescence to senescence [48]. PS has been reported to alter cytokine levels. Coussons-Read et al. [49] reported that stress-related neural immune R4  interactions may contribute to pregnancy complications and poor outcome. Collier et al. [50] found that PS changed typical proinflammatory cytokines including tumor necrosis factor (TNF)-α, and interleukin (IL)-6. As mentioned above, JKSQW recovered the dysfunction of kidney due to fear from earthquake, which could be supported by gene profile experiment outcome. In other words, cytokine conduction pathways, for example, JAK/STAT are involved in the prenatal kidney deficiency, and key molecules like Socs-2 and Socs-4 are the regulating targets of Chinese medicine treatment. The underlying mechanism that JKSQW improves development and behavior might attribute to the upregulation of Socs-2 and Socs-4 which suppress the pathway of JAK/STAT, resulting in reduction certain cytokines' secretion. diabetes is considered as Xiao-ke in Chinese medicine, whose major pattern is kidney deficiency. JKSQW plays an important role in the composition of prescriptions treating Diabetes in Chinese medicine [51]. Promisingly, our findings revealed insulin related pathways were involved in the outcome of herbal intervention in HG, supporting the hypnosis that JKSQW recovery the dysfunction of kidney. Four genes (Irf7, Ninj2, Plxnc1, and Isca1) were validated with RT-PCR, showing a favorable match (75%) between the gene expression profile chip and RT-PCR result. It is reported that all elements of IFN responses, whether the systemic production of IFN in innate immunity or the local action of IFN from plasmacytoid dendritic cells in adaptive immunity, are under the control of Irf7 [52]. Hannah et al. [53] reported that induction of pattern recognition receptors (PRRs; Tlr7 and Rig-I), expression of antiviral genes (Myd88, Visa, Jun, Irf7, Ifnbeta, Ifnar1, Jak2, Stat3, and Mx2), and production of Mx protein was elevated in the lungs of intact females compared with intact males. Ninjurin2 (Ninj2) is a transmembrane protein that mediates cell-to-cell and cell-to-extracellular matrix interactions during development, differentiation, and regeneration of the nervous system [54]. Recently, Ninj2 was reported to be a vascular susceptibility gene and associated with Alzheimer's disease risk [55].
In conclusion, together with our own recent data, the findings of this body of work demonstrate the earthquake as a prenatal stressor during the pregnancy could negatively retard the body and nervous system development, and Chinese herbal remedy could correct the retardation, which could attribute to neurohormones alteration and altered gene expression profile. The gene pathways involved have been tied to signaling pathway, long-term potentiation, ubiquitin mediated proteolysis, and long-term depression relating to disruptions from prenatal stress; Jak-STAT signaling pathway could play a key role in improving the function of JKSQW. This study demonstrates that negatively prenatal experiences have the ability to significantly retard

HG ESG
Color key and histogram   Figure 6: RT-PCR validation of the selected four genes from gene expression profile chips, that is, Irf7, Ninj2, and Plxnc1, and Isca1. ΔΔCt < 0 indicates the target genes were hyperexpressed in ESG/HG comparing with CG while ΔΔCt > 0 indicates the target genes were hypoexpressed in ESG/HG comparing with CG. FC > 2 indicates the target genes were hyperexpressed in ESG/HG comparing with CG while FC < 0.5 indicates the target genes were hypoexpressed in ESG/HG comparing with CG.
offspring developmental and immunity trajectories, which can be corrected by Chinese herbal remedy.