| Depression is a kind of common chronic mental disease with complex pathogenesis, which has not been fully clarified until now. With the development of the theory of fetal origin of adult disease, more and more studies have confirmed that some reasons of mental illness can be traced back to the fetus stage. During fetal stage maternal stress caused by a variety of adverse environmental conditions can lead to increased levels of glucocorticoid hormone(GCs) in the fetus. And excessive prenatal GCs exposure can program the expression of some key genes in the brain, which increase risk of depression, anxiety and other mental disorders in adult. In addition, clinic treatment for pregnant women who are at risk of preterm delivery usually given dexamethasone(DEX), a kind of synthetic glucocorticoid, to promote fetal lung maturation, reduce morbidity of respiratory distress syndrome and premature mortality. However, epidemiological studies have found that exposure to excessive synthetic GCs during late pregnancy greatly increased the risk of mental illness, emotional disorder problems in the adult offspring. What is the mechanism of excessive GCs exposure during later period of pregnancy have the effect on the depression-like behavior of the offspring? Dose this influence can been transmitted cross generationalion? And what is the mechanism? It is not very clear at present.Therefore, in the first part of this paper, we first construct excessive GCs exposure rat model during late pregnancy. Then we used sucrose consumption test, forced swimming test, tail suspension test and open field test to detect the behavior changes of the adult F1 generation offspring. After finished behavioral tests, some adult F1 generation were mated to produce F2 generation offspring, and behavior changes was detected again. At the same time, we used high-throughput genome-wide expression profiling to screen key molecules in brain of this model. Meanwhile, we used molecular biology technology to explore the mechanism of depression-like behavior and transgenerational transmission caused by excessive GCs exposure during late gestation.Clinical studies have confirmed that women mood disorders such as anxiety and depression rapid increase in the menstrual period which with sharp decline level of estrogen. At the same time, the menopausal women’s depression score was significantly negatively correlated with the level of plasma estradiol, while estrogen replacement therapy can improve the symptoms of mood disorders. It suggests that depression is closely related to serum estrogen level. At present, both clinical and animal experiments have confirmed that estrogen is involved in regulating emotional response, but its mechanism has not been fully clarified. Therefore, in the second part of this paper, we constructed estrogen deficient animal model by ovariectomized operation, which was used to observe the effect of estrogen deficiency on animal depression-like behavior. Then, we explored the effects of estradiol, estrogen receptor agonist, and inflammasome inhibitor on the depression-like behavior of ovariectomized mice. On the basis of this study, we tried to explore the molecular mechanism of the anti-depression effect of estrogen by using molecular biology method. The main results are as follows:Part one: The mechanisms involved in prenatal glucocorticoids exposure induced depression-like behavior in offsprings.Ⅰ. Depression-like behavior and transgenerational transmission caused by excessive GCs exposure during last period of pregnancyFirst, we constructed excessive GCs exposure rat model during late pregnancy. Pregnant F0 SD rats were abdominal subcutaneous injected with 0.1ml DEX solution(0.1mg/kg) on GD14-GD21 of pregnancy, the control group injected with the same amount of saline until birth. Then we used sucrose consumption test, forced swimming test, tail suspension test and open field test to detect the behavior changes of the adult F1 generation offspring. After finished behavioral tests, some adult F1 generation were mated to produce F2 generation offspring, and behavior changes was detected again.1. Prenatal DEX exposure leads to increased depression-like behavior in adult F1 offspringBehavioral experiment results of adult F1 offspring showed that: Prenatal DEX exposure induced depression-like behavior in male and female F1 offspring as evidenced by reduced sucrose consumption, increased immobility time in FST and TST and reduced the distance traveled in center as well as number of crossing squares in the OFT compared to the control F1 offspring, there was no gender difference.2. Transgenerational transmission of depression-like behavior induced by prenatal DEX exposureF2 males and females in Con♂/DEX♀ and DEX♂/DEX♀ groups showed depression-like behavior including reduced sucrose consumption, increased immobility time and reduced distance traveled in center as well as the number of crossing squares. In contrast, F2 males and females in DEX♂/Con♀ group did not show a significant change in TST and OFT but only showed decreased sucrose consumption. Thus, our study demonstrate that depression-like behavior is across generations induced by maternal DEX exposure in F0 and transgenerational depression-like behavior in F2 is predominantly dependent on maternal line.Ⅱ.The mechanisms involved in prenatal glucocorticoids exposure on depression-like behavior in offspring and transgenerational transmission1. Prenatal DEX exposure increased CRH and CRHR1 expression in hippocampus of F1 offspring(1) Microarray analysis to compare the gene expression profile in hippocampus of F1 offspringThe results showed that,among the regulated genes, the expression of CRH, serotonin(5-HT) receptor 5-HT1 A, olfactory receptor 840(Olr840), Olr232, aldehyde dehydrogenase 1 family member A2(Aldh1a2) and ubiquitin D(Ubd) was changed in all arrays in the prenatal DEX exposure group which related to brain function. Using Q-PCR, we confirmed that CRH m RNA was significantly increased in both male and female F1 DEX offspring. However, m RNA expression of Aldhla2, Olr840, Olr232 and Ubd showed inconsistent changes in male and female F1 DEX offspring. We indicated that, Aldhla2, Olr840, Olr232 and Ubd are not the key genes of the offspring depression-like behavior induced by prenatal DEX exposure.(2) Prenatal DEX exposure increased CRH and CRHR1 expression in hippocampus of F1 offspringReal-time PCR and Western blot results showed that CRHR1 m RNA and protein levels in hippocampus were significantly increased in both male and female F1 DEX offspring, whereas CRHR2 m RNA and protein expression was not significantly differed between F1 DEX and control rats. In addition, CRH and CRHR1 but not CRHR2 m RNA expression was increased in F1 embryos(E18) of either sex.2. High expression of CRHR1 in hippocampus lead to depression-like behavior(1) CP154526, CRHR1 antagonist, can improve depression-like behavior induced by prenatal DEX exposureF1 DEX rats at one-day old received CRHR1 antagonist CP154526 treatment for a week. These rats at 9-week old showed improvement of depression-like behavior with no significant sex difference in anhedonia, despair and locomotor activity compared with those with vehicle injection.(2) Overexpression of CRHR1 in hippocampus lead to depression-like behaviorLentiviral vectors carrying the CRHR1 gene were introduced into the hippocampus of normal adult male rats four weeks before behavioral tests. Initially we confirmed that i.h. injection of lentiviral CRHR1 gene expression vectors resulted in an increase in CRHR1 expression. Next, we found that injection of lentiviral CRHR1 gene expression vectors induced depression-like behavior including reduced sucrose consumption, increased immobility time in the TST and reduced distance traveled in center as well as number of crossing squares in the OFT. Suggesting that elevated CRH/CRHR1 signaling is a key contributor to prenatal DEX exposure induced depression-like behavior in offspring.3. CRH and CRHR1 in F1 offspring can be transmitted to F2 generation mainly through maternal lineReal-time PCR and Western blot results showed that CRH and CRHR1 in hippocampus was significantly upregulated in F2 E18 and adult offspring in Con♂/DEX♀ and DEX♂/DEX♀ groups but not in DEX♂/Con♀ group. In contrast, CRHR2 did not have significant changes in the hippocampus in F2 generations. Thus, we conclude that elevated expression of CRH and CRHR1 in F1 offspring due to prenatal DEX exposure can be transmitted to the F2 generation mainly through the maternal line.4. The mechanisms involved in prenatal glucocorticoids exposure on CRHR1 expression and transgenerational transmission(1) Prenatal DEX exposure decreased methylation level of CpG site within CRHR1 promoter in the hippocampus of F1 offspringWe examined global methylation and the methylation status of specific Cp G island promoter regions of CRH and CRHR1 genes in the hippocampus of adult F1 male rats. Prenatal DEX exposure resulted in a significant decrease in global methylation in F1 offspring. A significant decrease in methylation frequency of the-833 and +13 Cp G sites in CRHR1 promoter was found in F1 DEX rats. However, there was no significant change in methylation frequency of Cp G sites in the CRH promoter of F1 DEX offspring. Thus, this result suggests that hypomethylation of the CRHR1 promoter region is likely a potential mechanism responsible for elevated hippocampus CRHR1 gene expression.(2) Decreased methylation level of Cp G site within CRHR1 promoter can be transmitted to F2 generation mainly through maternal lineThe results showed that, global methylation in F2 offspring from Con♂/DEX♀ and DEX♂/DEX♀groups was significant decreased. Decreased methylation frequency of the-732,-671,-527,-128,-76 and-10 Cp G sites in CRHR1 promoter were found in F2 offspring deprived from female F1 DEX offspring. However, an increase in global methylation was found in female F2 rats derived from DEX♂/Con♀ group. These results suggest that decreased methylation level of Cp G site within CRHR1 promoter can be transmitted to F2 generation mainly through maternal line.(3) DEX increase CRHR1 expression in hippocampal slice via inhibition DNA methylationReal-time PCR and Western blot results showed that treatment of cultured hippocampal slices with DEX caused an increase in CRH and CRHR1 expression in a dose-dependent manner, whereas DEX treatment had no effect on CRHR2 m RNA expression. DEX treatment resulted in a significant decrease in global methylation. DEX-induced CRHR1 expression was reversed by the presence of DNA-methyltransferase agonist 3ABA whereas CRH expression induced by DEX did not affected by 3ABA treatment. These results indicated that DEX-induced expression of the CRHR1 gene dependent on decreased DNA methylation.Bioinformatics analysis showed that +13 Cp G site is located within the HSP binding site of CRHR1 promoter region. To test the importance of this region in CRHR1 promoter activity, we transfected plasmids expressing a luciferase reporter driven by either CRHR1 promoter containing HSP binding site or CRHR1 promoter containing specific mutation of HSP binding site around +13 Cp G site to HT-22 cells,. We found that mutation of the +13 Cp G site substantially decreased CRHR1 promoter activity by nearly 70% in HT-22 cells. Taken together, our study demonstrates that decreased methylation of +13 Cp G site in CRHR1 promoter region is important for prenatal s GC exposure-induced elevated CRHR1 gene expression.(4) Transgenerational inheritance of serum corticosterone in F1 DEX group can lead to low methylation level of hippocampusThe results of serum corticosterone level in F1 and F2 offspring showed that, serum corticosterone level in E18 F1 and F2 offspring was significant increased. Of note, we found that circulating corticosterone levels of F2 fetuses were only significantly induced in Con♂/DEX♀ and DEX♂/DEX♀ groups but not in DEX♂/Con♀ group, indicating that prenatal DEX exposure-mediated transgenerationally elevated fetal corticosterone levels is dependent on the maternal line. Treatment of cultured hippocampal slices with corticosterone caused an increase in CRH and CRHR1 expression in a dose-dependent manner, whereas corticosterone treatment had no effect on CRHR2 m RNA expression. In addition, we also found that corticosterone treatment resulted in a significant decrease in global methylation. These results indicated that increased CRH and CRHR1 gene expression and decreased DNA methylation in F2 offspring.5. The mechanism of high serum corticosterone level in F2 offspring(1) DEX exposure resulted in long lasting HPA hyperactivityThe results of serum corticosterone level showed that maternal circulating corticosterone levels were only significantly increased in the pregnant F1 DEX female mated with either F1 control or DEX male. CRH m RNA and protein expression in hypothalamus was increased whereas GR m RNA and protein expression in hippocampus was decreased in F1 DEX rats. The above results indicate that prenatal DEX exposure resulted in long lasting HPA hyperactivity in females, thereby causing persistently increased GC level during pregnancy.(2) Increased expression of 11β-HSD1 in F1 female placenta during pregnancyReal-time PCR and Western blot results showed that 11β-HSD1 m RNA and protein level was significantly upregulated in the placentas of male and female F2 fetus(GD18) in Con♂/DEX♀ and DEX♂/DEX♀ groups although it was decreased in male placentas of DEX♂/Con♀ group. In contrast, placental 11β-HSD2 m RNA and protein level did not significantly differ among all the groups.Part two: The mechanisms involved in depression-like and anxiety-like behavior induced by estrogen deficiency.1. Depression-like and anxiety-like behavior induced by estrogen deficiency is reversed by ERβ agonistFirst, OVX mouse model was constructed. One week after OVX surgery, OVX mice were randomly divided into OVX group, OVX-E2(10μg/kg) group, OVX-E2(30μg/kg) group, OVX-PPT(10μg/kg) group, OVX-PPT(30μg/kg) group, OVX-DPN(10μg/kg) group, OVX-DPN(30μg/kg) group. Abdominal subcutaneous injection of 0.1ml E2, estrogen receptor agonist PPT and DPN, Sham group was injected with sesame oil, After 4-week treatment with E2 and agonists of ERs, the behavioral tests were performed.(1) ERβ agonist DPN can ameliorate depression-like behavior in OVX miceOVX mice showed a significant decrease in the percentage of sucrose consumption and a significant increase in immobility time as compared to Sham mice. OVX mice received E2 replacement(10 or 30 μg/kg/day) showed an increase in sucrose preference compared with OVX-vehicle mice. Treatment of OVX mice with DPN(30 μg/kg/day) also significantly increased sucrose preference. OVX mice with E2 and DPN replacement at 30 μg/kg/day showed a decrease in immobility time as compared to vehicle-treated OVX mice. E2 and DPN replacement at 10 μg/kg/day had no significant effect on immobility time in TST. PPT replacement had no effect on the above behavior in OVX mice.(2) ERβ agonist DPN can ameliorate anxiety-like behavior in OVX miceIn EZM, OVX mice also showed a significant decrease in the time spent in the open arms and number of entries into the open arms as compared to Sham mice. In OFT, OVX mice showed a significant decrease in distance traveled in center, number of crossing squares and the time in the central compared with Sham group. OVX with E2 and DPN replacement(30 μg/kg/day) showed an increase in the time spent in the open arms and number of entries into the open arms as compared to vehicle-treated OVX group in EZM. E2 treatment at lower dosage(10 μg/kg/day) could improved the time spent in the open arms while DPN treatment at lower dosage(10 μg/kg/day) improved the number of entries into the open arms. In OFT, OVX with E2 and DPN replacement(30 μg/kg/day) showed a significant increase in distance traveled in center, number of crossing squares and the time in the central as compared with vehicle-treated OVX group. E2 and DPN treatment at lower dosage(10 μg/kg/day) had no significant effect on the behavior in OFT. Administration of PPT had no significant effect on the above behavior in OVX mice.2. ERβ agonist DPN can ameliorate inflammation in hippocampus of OVX mice(1) Ovariectomy induced IL-1β and IL-18 production in hippocampus is reversed by ERβ agonistELISA analysis showed that the levels of IL-1β and IL-18 in hippocampus were significantly increased in OVX mice compared with those in Sham group. With administration of E2(30 μg/kg/day) and DPN(30 μg/kg/day), the levels of IL-1β and IL-18 were significantly decreased in OVX mice as compared vehicle-treated OVX group. PPT treatment had no effect on IL-1β and IL-18 levels. OVX mice showed higher level of IL-1β and IL-18 m RNA expression than Sham group. E2(30 μg/kg/day) and DPN(30 μg/kg/day) replacement reversed ovariectomy-induced increase of IL-1β and IL-18 m RNA expression.We therefore examined the levels of IL-1β and IL-18 in PFC and amygdala in OVX mice and found that there was no significant difference in IL-1β and IL-18 levels in PFC and amygdala between OVX group and Sham group. PPT and DPN treatment did not affect IL-1β and IL-18 levels in these brain regions in OVX mice.(2) Ovariectomy leading to NLRP3 inflammasome activation in hippocampus is reversed by ERβ agonistReal-time PCR and Western blot results showed that NLPR3 m RNA and protein expression were significantly increased in OVX mice as compared to Sham group. The level cleaved caspase-1 P10(active caspase-1) protein were also significantly increased in OVX mice as compared to Sham group. However, ASC protein expression was not significantly changed upon to ovariectomy. With E2(30 μg/kg/day) or ERβ agonist DPN(30 μg/kg/day) treatment for four weeks, NLPR3 expression and caspase-1 P10 level were significantly decreased in OVX mice. PPT treatment did not affect the level of NLPR3 and caspase-1 P10. Mature IL-1β and IL-18 levels were significantly increased in OVX group. Moreover, pro-IL-1β and pro-IL-18 protein levels were also significantly increased in OVX group. With E2(30 μg/kg/day) or ERβ agonist DPN(30 μg/kg/day) treatment, the levels of pro-IL-1β, pro-IL-18, IL-1β and IL-18 were significantly decreased as compared with vehicle-treated OVX mice.(3) The increase in TLR-2 and TLR-4 expression and NF-κB signaling activation by ovariectomy are reversed by ERβ agonistWestern blot results showed that TLR-2 and TLR-4 protein expression were significantly increased in OVX mice. My D88 was also increased in OVX mice. 30 μg/kg/day E2 and DPN replacement reversed the increase of TLR-2, TLR-4 and My D88 expression in OVX mice. The level of phosphorylated p65(p-p65) was significantly increased in OVX mice compared with Sham group. 30 μg/kg/day E2 and DPN replacement decreased the level of p-p65 in OVX mice, whereas PPT had no effect on p-p65 level.(4) Ovariectomy induction of P2X7 R and CD11 b expression in hippocampus is reversed by ERβ agonistWestern blot results showed that P2X7 R protein expression was significantly increased in OVX mice. Administration of E2(30 μg/kg/day) and DPN(30 μg/kg/day) but not PPT(30 μg/kg/day) reduced P2X7 R protein expression in OVX mice. OVX mice exhibited increased expression of microglia cell marker CD11 b protein in hippocampus as compared to Sham group. E2(30 μg/kg/day) or DPN(30 μg/kg/day) replacement but not PPT replacement reduced the protein expression of CD11 b in OVX mice.3. VX-765 reverse depression-like and anxiety-like behavior and inflammation in OVX mice(1) VX-765 reverses depression-like and anxiety-like behavior induced by ovariectomyAdministration of VX-765 ameliorated depression-like and anxiety-like behavior in OVX mice. VX-765(50 mg/kg/day) significantly increased percentage of sucrose consumption in OVX mice as compared to vehicle-treated OVX group. OVX mice with VX-765 treatment at the dosage of 25 or 50 mg/kg/day showed decreased immobility time in TST compared to those with vehicle treatment. Treatment with VX-765 at the dosage of 25 or 50mg/kg/day could increase the time spent in the open arms and number of entries into the open arms in OVX mice. In OFT, 50 mg/kg/day VX-765 treatment reversed decreased exploring behavior including the distance traveled in center, number of crossing squares and time spent in center.(2) VX-765 reverses ovariectomy-induced IL-1β, IL-18, NF-κB activation and CD11 b in hippocampusWestern blot results showed that with administration of VX-765(25 mg/kg/day and 50 mg/kg/day), the mice showed decreased levels of mature IL-1β and IL-18 compared to those with vehicle treatment. Pro-IL-18 level in OVX mice was decreased by VX-765 treatment at dosage of 50 mg/kg/day. ELISA analysis also showed that VX-765 treatment(25 mg/kg/day and 50 mg/kg/day) significantly decreased the content of IL-1β and IL-18 in hippocampus of OVX mice. VX-765 treatment(50 mg/kg/day) decreased p-p65 and CD11 b level in hippocampus of OVX mice. Treatment of OVX mice with VX-765 at 25 mg/kg/day significantly reduced CD11 b protein level in hippocampus.Conclusion:1. Prenatal GCs exposure can lead to depression-like behavior in F1 offspring which can be transmitted to F2 generation mainly through maternal line. The mechanism may be related to the low methylation level in hippocampus which lead to the high expression of CRHR1 in F1 offspring.2. Prenatal DEX exposure results in down regulation of GR and long lasting HPA hyperactivity in F1 female and upregulated placental 11β-HSD1 gene expression during pregnancy, subsequently leading to elevated fetal circulating GCs and causing subsequent epigenetic modification of fetal hippocampus CRHR1 and eventual depression behavior in second generation.3. Estrogen and ERβ agonist DPN can reverse depression-like and anxiety-like behavior in OVX mice. The mechanism may be relative to inhibition of NLRP3 inflammasome, thereby inhibit microglia activation and reduce the expression of IL-1β and IL-18 in hippocampus.4. OVX activated P2X7 R, TLR-2, TLR-4 and its downstream signal molecular NF-κB, My D88. NLRP3 inflammasome inhibitor VX-765 can inhibit proinflammatory cytokine IL-1β and IL-18 expression in hippocampus, and then improve depression-like and anxiety-like behavior. |
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