Modulation of the suppressive effect of corticosterone on adult rat hippocampal cell proliferation by paroxetine

Objective The literature has shown that cognitive and emotional changes may occur after chronic treatment with glucocorticoids. This might be caused by the suppressive effect of glucocorticoids on hippocampal neurogenesis and cell proliferation. Paroxetine, a selective serotonin reuptake transporter, is a commonly used antidepressant for alleviation of signs and symptoms of clinical depression. It was discovered to promote hippocampal neurogenesis in the past few years and we wanted to investigate its interaction with glucocorticoid in this study. Methods Adult rats were given vehicle, corticosterone, paroxetine, or both corticosterone and paroxetine for 14 d. Cell proliferation in the dentate gyrus was quantified using 5-bromo-2-deoxyuridine （BrdU） immunohistochemistry. Results The corticosterone treatment suppressed while paroxetine treatment increased hippocampal cell proliferation. More importantly, paroxetine treatment could reverse the suppressive effect of corticosterone on hippocampal cell proliferation. Conclusion This may have clinic application in preventing hippocampal damage after glucocorticoid treatment.

Effects of Corticosterone, cAMP, cGMP, Ca^(2+), and Protein Kinase C on Apoptosis of Mouse Thymocytes Induced by X-ray Irradiation

Objective To observe the effects of signal factors of corticosterone （CS）, cAMP, cGMP, Ca^2＋ and protein kinase C （PKC） on lymphocyte apoptosis in mouse thymus induced by X-rays of 4 Gy in vitro. Methods The DNA lyric rate for thymocytes was measured by fluomspectrophotometry. Results The DNA lyric rate for thymocytes 4-8 hours after irradiation with 2-8 Gy was significantly higher than that in the control （P〈0.01）. As compared with the control, the DNA lyric rate for thymocytes treated with 0.01 μnol/L CS （P〈0.01）, 50 ng/mL cAMP （P〈0.01）, 0.05-0.4 μg/mL ionomycin （Iono, P〈0.05 or P〈0.01） or 0.05-0.4 ng/mL phorbol myristate acetate （PMA, P〈0.05 or P〈0.01）, respectively, was significantly increased, while the rate for thymocytes treated with 50 ng/mL cGMP was not significantly increased. The DNA lyric rate for thymocytes treated with 0.01 μmol/L CS （P〈0.01）, 50 ng/mL cAMP （P〈0.01）, 0.2 and 0.4 μg/mL Iono （P〈0.05）, and 0.2 and 0.4 ng/mL PMA （P〈0.05） plus 4-Gy irradiation, respectively, was significantly higher than that treated with single 4-Gy irradiation, while the rate for thymocytes treated with 50 ng/mL cGMP plus 4-Gy irradiation was not increased. When both 0.4 I.tg/mL Iono and 0.4 ng/mL PMA acted on the thymocytes, the DNA lyric rate for thymocytes was significantly higher than that in the control （P〈0.01）, the DNA lytic rate for thymocytes treated with both 0.4 μg/mL Iono and 0.4 ng/mL PMA plus 4-Gy irradiation was significantly higher than that treated with single 4-Gy irradiation （P〈0.05）, but was Iono plus 4-Gy irradiation or 0.4 ng/mL PMA plus 4-Gy irradiation. can promote thymocyte apoptosis induced by larger dose X-rays. not significantly higher than that treated with 0.4 μg/mL Conclusion CS, cAMP, Ca^2＋, and PKC signal factors can promote thymocyte apoptosis induced by larger dose X-rays.

Active fraction combination from Liuwei Dihuang decoction(LW-AFC) ameliorates corticosterone-induced long-term potentiation impairment in mice in vivo

Liuwei Dihuang decoction(LW), a classic formula in traditional Chinese medicine(TCM), has been used for nearly one thousand years for various diseases with characteristic features of kidney yin deficiency. LW consists of 6herbs including Dihuang[prepared root of Rehmannia glutinosa(Gaertn) DC], Shanyao(rhizome of Dioscorea polystachya Turcz), Shanzhuyu(fruit of Cornus officinalis Siebold Zucc), Mudanpi(root bark of Paeonia × suffruticosa Andrews),Zexie(rhizome of Alisma plantago-aquatica L) and Fuling(scleorotia of Wolfiporia extensa(Peck) Ginns)LW-active fraction combination(LW-AFC) is extracted from LW, it is effective for the treatment of kidney yin deficiency in many animal models. There are 3 fractions in LW-AFC, a polysaccharide fraction(LWB-B), a glycoside fraction(LWD-b) and an oligosaccharide fraction(CA-30). Our previous results indicate that LW-AFC has similar pharmacological effects to LW, modulating the balance of the NIM network. LW-AFC has positive effects in many animal models of kidney deficiency or disturbance of the NIM network. LW-AFC could improve the cognitive ability in Alzheimer′s disease(AD) animal models(APP/PS1, SAMP8), where modulating immune function and balancing the NIM network may play an important role in its cognition improving effects. Our study also showed that LW-AFC had protective effects on stress-induced disturbances of the NIM network. However, the underlying mechanisms remain elusive and need further investigation. OBJECTIVE This study evaluated the effects of LW-AFC and the active fractions(polysaccharide, LWB-B;glycoside, LWD-b;oligosaccharide,CA-30) on corticosterone(Cort)-induced long-term potentiation(LTP) impairment in vivo. METHODS LTP was used to evaluate the synaptic plasticity. LW-AFC was orally administered for seven days. The active fractions were given by either chronic administration(ig, ip, 7 d) or single administration(icv, ig, ip). Cort was injected subcutaneously 1 h before the high-frequency stimulation(HFS) to induce LTP impairment. Moreover, in order to research on the possible effective pathways, an antibiotic cocktail and an immunosuppressant were also used. RESULTS Chronic administration(ig) of LW-AFC and its three active fractions could ameliorate Cort-induced LTP impairment. Single administration(icv, ig, ip) of any of the active fractions had no effect on Cort-induced LTP impairment, while chronic administration(ig, ip) of LWB-B or LWD-b showed positive effects against Cort. Interestingly, CA-30 only showed protective effects via ig administration,and there was little effect when CA-30 was administered ip In addition, when the intestinal microbiota was disrupted by application of the antibiotic cocktail, CA-30 showed little protective effects against Cort. The effects of LW-AFC were also abolished when the immune function was inhibited. In the hippocampal tissue, Cort treatment increased corticosterone and glutamate, and LW-AFC could inhibit the Cort-induced elevation of corticosterone and glutamate;there was little change in D-serine in Cort-treated animals, but LW-AFC could increase the D-serine levels. CONCLUSION LW-AFC and its three active fractions could ameliorate Cort-induced LTP impairment. Their protective effects are unlikely by a direct way, and immune modulation might be the common pathway. CA-30 could protect LTP from impairment via modulating the intestinal microbiota. Decreasing corticosterone and glutamate and increasing D-serine in the Cort-treated animals’ hippocampal tissue might be one of the mechanisms for the neural protective effects of LW-AFC. Further study is needed to understand the underlying mechanisms.

Involvement of nuclear factor-kappa B on corticosteroneinduced rat Leydig cell apoptosis

Aim： To investigate the activation of nuclear factor-kappa B （NF-kappa B） and its function in glucocorticoid-induced Leydig cell apoptosis. Methods： The Leydig cells were isolated from male Sprague-Dawley rats （90 days of age） and were incubated with corticosterone （CORT, glucocorticoid in rat） for 6 h, 12 h and 24 h, respectively. The P65 subunit of NF-kappa B （NF-kappa B/P65） in nuclei and the inhibitor of NF-kappa B （Ikappa B） in cytoplasm were analyzed by Western-blotting. The Leydig cells were treated with anti-Fas antibody for 3 h followed by Western blotting to assay the changes of NF-kappa B/P65 in nuclei and in cytoplasm. The role of NF-kappa B in CORT- induced Leydig cell apoptosis was evaluated by observing the effects of NF-kappa B/P65 overexpression and inhibiting activation of NF-kappa B by 100 μmol/L Pyrrolidine dithiocarbamate （PDTC） on this apoptosis. Results： The treatment of Leydig cells with CORT increased the levels of NF-kappa B/P65 in nuclei and decreased the levels of Ikappa B in cytoplasm. Following the Leydig cells were treated with anti-Fas antibody, the levels of NF-kappaB/P65 was increased in nuclei and decreased in cytoplasm. The CORT-induced Leydig cell apoptosis was inhibited by overexpressed NF-kappaB/P65 and was enhanced by incubation with PDTC. Conclusion： NF-kappa B is activated by increased FasL/Fas in CORT-induced Leydig cell apoptosis. NF-kappa B may play an anti-apoptotic role in this apoptosis.

Effects of capture and captivity on plasma corticosterone and metabolite levels in breeding Eurasian Tree Sparrows

Background: Bringing free-living animals into captivity subjects them to the stress of both capture and captivity, leading to the alteration of normal physiological processes and behaviors through activation of the hypothalamic– pituitary–adrenal axis. In free-living birds, although elevated plasma corticosterone (CORT) is an important adaptation regulating physiological and behavioral responses during the process of capture and captivity stress, little information is currently available on the effects of such stress on plasma metabolite levels. Methods: We examined the effects of immediate capture and 24-h captivity on body mass, body condition, plasma CORT, and metabolite levels including glucose (Glu), triglyceride (TG), total cholesterol (TC), uric acid (UA), in breeding Eurasian Tree Sparrows (Passer montanus). Results: CORT and Glu levels were increased significantly by the stress of capture, whereas TC and UA levels decreased. Body mass, body condition declined notably after 24 h in captivity, but CORT, Glu, and UA levels increased. Furthermore, male sparrows had lower TG levels after both capture and captivity than those of females. The relationships between plasma CORT and metabolite levels varied between sexes. Conclusions: Our results revealed that the metabolic status of Eurasian Tree Sparrows could be dramatically altered by capture and captivity. Monitoring the dynamic effects of both capture and captivity on plasma CORT, metabolite levels in a free-living bird contributes to a better understanding of the stress-induced pathways involved in sexdependent energy mobilization.

Geniposide protects human neuroblastoma SH-SY5Y cells against corticosterone-induced injury

In vitro cultured human neuroblastoma SH-SY5Y cells were pretreated with 50 or 5 ug/mL geniposide for 12 hours and exposed to 400 umol/L corticosterone. Corticosterone exposure in cultures not pretreated with geniposide resulted in inhibited cell growth, reduced cell survival, and increased P53 and P21 protein expression. However, in geniposide pretreated SH-SY5Y cells, cell viability and the number of cells in the G2 phase of the cell cycle were significantly increased, P21 and P53 protein expression was reduced, and cell apoptosis was inhibited following corticosterone exposure. These results indicate that geniposide can protect SH-SY5Y cells against high-dose corticosterone-induced injury.

NFAT2 is implicated in corticosterone-induced rat Leydig cell apoptosis

Aim： To investigate the activation of the nuclear factor of activated T cells （NFAT） and its function in the corticosterone （CORT）-induced apoptosis of rat Leydig cells. Methods： NFAT in rat Leydig cells was detected by Western blotting and immunohistochemical staining. Cyclosporin A （CsA） was used to evaluate potential involvement of NFAT in the CORT-induced apoptosis of Leydig cells. Intracellular Ca^2＋ was monitored in CORT-treated Leydig cells using Fluo-3/AM. After the Leydig cells were incubated with either CORT or CORT plus CsA for 12 h, the levels of NFAT2 in the nuclei and in the cytoplasm were measured by semi-quantitative Western blotting. The role of NFAT2 in CORT- induced Leydig cell apoptosis was further evaluated by observing the effects of NFAT2 overexpression and the inhibition of NFAT2 activation by CsA on FasL expression and apoptosis. Results： We found that NFAT2 was the predominant isoform in Leydig cells. CsA blocked the CORT-induced apoptosis of the Leydig cells. The intracellular Ca^2＋ level in the Leydig cells was significantly increased after the CORT treatment. The CORT increased the level of NFAT2 in the nuclei and decreased its level in the cytoplasm. CsA blocked the CORT-induced nuclear translocation of NFAT2 in the Leydig cells. Both CORT-induced apoptosis and FasL expression in the rat Leydig cells were enhanced by the overexpression of NFAT2 and antagonized by CsA. Conclusion： NFAT2 was activated in CORT-induced Leydig cell apoptosis. The effects of NFAT2 overexpression and the inhibition of NFAT2 activation suggest that NFAT2 may potentially play a pro-apoptotic role in CORT-induced Leydig cell apoptosis through the up-regulation of FasL.

Effect of relative humidity at either acute or chronic moderate temperature on growth performance and droppings' corticosterone metabolites of broilers

The present study aimed to investigate the effect of relative humidity(RH) at either acute or chronic moderate ambient temperature(Ta) on growth performance and droppings' corticosterone metabolites of broilers.Two experiments were conducted: effect of RH(35,60 or 85%) on average daily feed intake(ADFI) and droppings' corticosterone metabolites at acute(1 d: 20–26 or 31–20°C,26 or 31°C for 6 h d–1 at 10:00–16:00) moderate Ta(experiment 1) and effect of RH(35,60 or 85%) on growth performance and droppings' corticosterone metabolites at chronic(step-wisely increasing temperature by 3°C every 3 d from 20 to 32°C within 15 d: 20–23–26–29–32°C) moderate Ta(experiment 2).Droppings were collected at the 2,4,6,8,and 22 h after Ta-RH controlled in experiment 1 and at the 2,4,6,and 22 h after Ta controlled to 32°C in experiment 2.The results showed that: 1) In experiment 1,85% RH increased(P<0.05) the droppings' corticosterone metabolites at the 2,6,8,and 22 h and 35% RH increased(P<0.05) it at the 2 and 22 h compared to the 60% RH.Moreover,85% RH further increased(P<0.05) it compared to the 35% RH,however,no difference(P>0.05) was found in ADFI among the three RH groups at acute moderate 26°C; 35 and 85% RH increased(P<0.05) droppings' corticosterone metabolites at the 2,6,8 and 22 h and decreased(P<0.05) ADFI compared to the 60% RH,moreover,85% RH further increased(P<0.05) droppings' corticosterone metabolites and further decreased(P<0.05) ADFI compared to the 35% RH at acute moderate 31°C; and the average of droppings' corticosterone metabolites in the whole period had a negative correlation(P<0.02) with the ADFI.2) In experiment 2,85% RH increased(P<0.01) droppings' corticosterone metabolites only at the 2 h and decreased(P<0.02) ADFI and average daily gain(ADG) compared to the 60% RH,no difference(P>0.05) in droppings' corticosterone metabolites was found between the 35 and 60% RH,however,35% RH decreased(P<0.01) ADG compared to the 60% RH,and the average of droppings' corticosterone metabolites in the whole period also had a negative correlation(P<0.02) with ADFI and ADG.In conclusion,droppings' corticosterone metabolites could be used as a RH stress index and low and high RH,especially high RH,reduced growth performance possibly through inducing RH stress at moderate temperature.

Maternal betaine suppresses adrenal expression of cholesterol trafficking genes and decreases plasma corticosterone concentration in offspring pullets

Background:Laying hens supplemented with betaine demonstrate activated adrenal steroidogenesis and deposit higher corticosterone(CORT)in the egg yolk.Here we further investigate the effect of maternal betaine on the plasma CORT concentration and adrenal expression of steroidogenic genes in offspring pullets.Results:Maternal betaine significantly reduced(P<0.05)plasma CORT concentration and the adrenal expression of vimentin that is involved in trafficking cholesterol to the mitochondria for utilization in offspring pullets.Concurrently,voltage-dependent anion channel 1 and steroidogenic acute regulatory protein,the two mitochondrial proteins involved in cholesterol influx,were both down-regulated at m RNA and protein levels.However,enzymes responsible for steroid syntheses,such as cytochrome P450 family 11 subfamily A member 1 and cytochrome P450 family 21 subfamily A member 2,were significantly(P<0.05)up-regulated at m RNA or protein levels in the adrenal gland of pullets derived from betaine-supplemented hens.Furthermore,expression of transcription factors,such as steroidogenic factor-1,sterol regulatory element-binding protein 1 and c AMP response element-binding protein,was significantly(P<0.05)enhanced,together with their downstream target genes,such as 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase,LDL receptor and sterol regulatory element-binding protein cleavage-activating protein.The promoter regions of most steroidogenic genes were significantly(P<0.05)hypomethylated,although methyl transfer enzymes,such as AHCYL,GNMT1 and BHMT were up-regulated.Conclusions:These results indicate that the reduced plasma CORT in betaine-supplemented offspring pullets is linked to suppressed cholesterol trafficking into the mitochondria,despite the activation of cholesterol and corticosteroid synthetic genes associated with promoter hypomethylation.

Shuyusan-containing serum protects SH-SY5Y cells against corticosterone-induced impairment

The Chinese herb Shuyusan, whose main constituent is jasminoidin, has been shown to protect SH-SY5Y cells against corticosterone-induced damage. SH-SY5Y cells injured by 400 μmol/L cor- ticosterone were treated with 5 and 30 μg/mL Shuyusan-containing serum. Results revealed that Shuyusan-containing serum elevated the survival rate of SH-SY5Y cells, reduced Bax expression, increased Bcl-2 expression, markedly elevated brain-derived neurotrophic factor mRNA expression, and blocked cell apoptosis. Moreover, the effect of high-dose （30 μg/mL） Shuyusan-containing se- rum was more remarkable. Therefore, Shuyusan-containing serum appears to protect SH-SY5Y cells against corticosterone-induced impairment by adjusting the expression of apoptosis-associ- ated proteins and brain-derived neurotrophic factor. Moreover, high-dose Shuyusan-containing se- rum has a protective effect on high-dose corticosterone-induced impairment.

Chronic corticosterone disrupts the circadian rhythm of CRH expression and m^(6)A RNA methylation in the chicken hypothalamus

Background:Corticotropin-releasing hormone(CRH),the major secretagogue of the hypothalamic-pituitary-adrenal(HPA)axis,is intricately intertwined with the clock genes to regulate the circadian rhythm of various body functions.N6-methyladenosine(m^(6)A)RNA methylation is involved in the regulation of circadian rhythm,yet it remains unknown whether CRH expression and m^(6)A modification oscillate with the clock genes in chicken hypothalamus and how the circadian rhythms change under chronic stress.Results:Chronic exposure to corticosterone(CORT)eliminated the diurnal patterns of plasma CORT and melatonin levels in the chicken.The circadian rhythms of clock genes in hippocampus,hypothalamus and pituitary are all disturbed to different extent in CORT-treated chickens.The most striking changes occur in hypothalamus in which the diurnal fluctuation of CRH mRNA is flattened,together with mRNA of other feeding-related neuropeptides.Interestingly,hypothalamic m^(6)A level oscillates in an opposite pattern to CRH mRNA,with lowestm^(6)A level after midnight(ZT18)corresponding to the peak of CRH mRNA before dawn(ZT22).CORT diminished the circadian rhythm of m^(6)A methylation with significantly increased level at night.Further site-specific m^(6)A analysis on 3’UTR of CRH mRNA indicates that higher m^(6)A on 3’UTR of CRH mRNA coincides with lower CRH mRNA at night(ZT18 and ZT22).Conclusions:Our results indicate that chronic stress disrupts the circadian rhythms of CRH expression in hypothalamus,leading to dysfunction of HPA axis in the chicken.RNA m^(6)A modification is involved in the regulation of circadian rhythms in chicken hypothalamus under both basal and chronic stress conditions.

Caspase-3 activation is required for corticosteroneinduced rat Leydig cell apoptosis

Objective: Study on caspase-3 expression and its activation in CORT-mediated rat Leydig cell apoptosis. Methods: Caspase-3 protein activity, content and the timing of the onset of caspase-3 cleavage in relationship to CORT administration in rats was studied by Western blot. The timing of the onset of caspase-3 activity in Leydig cell was measured by the fluorescence. Results: Low molecular weight DNA fragments that are characteristic of apoptosis were evident in Leydig cells by 12 h of exposure to 100 nmol/L CORT in vitro and it was more pronounced at 24 h. Western blot analysis revealed that procaspase-3 was low in untreated Leydig cells and increased by 6 h of CORT administration. By 12 h, however, procaspase-3 was significantly reduced and the cleaved, active caspase-3 forms appeared and increased through 24 h. In the presence of a specific caspase inhibitor, Ac-DEVD-CHO, Leydig cell apoptosis was suppressed, corroborating the hypothesis that caspase-3 is involved in CORT-mediated cell death. Conclusion: Caspase-3 was implicated in CORT-mediated rat Leydig cell apoptosis.

The Role of Pineal Gland in Regulating Circadian Corticosterone Rhythm and Its Mechanism

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Urinary corticosterone and normetanephrine levels after voluntary wheel and forced treadmill running in the db/db mouse

Exercise is recommended for the treatment of type 2 diabetes because of its benefits on body weight and glycemic control. Our recent work using the db/db mouse, a model that mimics the phenotype of type 2 diabetes, demonstrated that forced treadmill training exerted detrimental effects on obesity, hyperglycemia and insulin resistance. We investigated whether this response is explained by increased corticosterone and norepinephrine secretion, measured as urinary byproducts, since these hormones are known to alter glucose homeostasis. Male db/db mice and lean littermates serving as controls, were assigned to sedentary, voluntary wheel, and forced treadmill training groups for a period of 5 weeks. After 5 weeks of treadmill running, db/db mice remained hyperglycemic compared to sedentary db/db mice and were hyperinsulinemic compared to db/db voluntary runners. Urine glucose and corticosterone levels were also highest in db/db treadmill runners compared to all groups. Urine normetanephrine levels, although lower in db/db mice compared to control mice, were increased after treadmill running. Our results indicate that treadmill running leads to perturbations in plasma levels of hormones associated with glucose homeostasis. A greater stress response may be invoked by treadmill training, worsening glycemic control in this model of type 2 diabetes.

Propofol increases in vitro proliferation of cultured rat hippocampal precursor cells inhibited by corticosterone

BACKGROUND： Previous studies have shown that propofol enhances proliferation of cultured hippocampal precursor cells in vitro and increases proliferation of cultured hippocampal precursor cells inhibited by corticosterone. Because gamma-aminobutyric acid A （GABA-A） receptor is the functional target for propofol, the proliferative effects of propofol are thought to take place through GABA-A receptor. OBJECTIVE： To determine whether propofol enhances proliferation of rat hippocampal precursor cells inhibited by corticosterone by upregulating expression of GABA-A receptor. DESIGN, TIME AND SETTING： A comparative, observational, in vitro experiment was performed at the Beijing Institute of Pharmacology and Toxicology from April 2005 to April 2006. MATERIALS： Propofol was purchased from AstraZeneca, italy; corticosterone was purchased from Sigma, USA; bicuculline was purchased from Alexis, Switzerland. METHODS： Hippocampal precursor cells were isolated from newborn Wistar rats and cultured in vitro. The second passage of precursor cells was grouped according to the various drugs added to the culture medium： 0.5 μmol/L propofol; 2.5 pmol/L propofol; 100 μmol/L corticosterone; 10 μmol/L bicuculline; 100 μmol/L corticosterone and 0.5 μmol/L propofol; 100 μmol/L corticosterone and 2.5 μmol/L propofol; 100 μmol/L corticosterone, 10 μmol/L bicuculline, and 0.5 μmol/L propofol; 100 μmol/L corticosterone, 10 μmol/L bicuculline, and 2.5 μmol/L propofol; 100 μmol/L corticosterone and 10 pmol/L bicuculline. The cells were cultured for 24 hours with medium containing the respective concentration of drug. The control group consisted of precursor cells absent of drug treatment. MAIN OUTCOME MEASURES： The MTT and ^3H-TdR incorporation assays were used to detect proliferative effects of propofol and bicuculline on cultured rat hippocampal precursor cells inhibited by corticosterone. Immunocytochemistry was used to detect GABA-A receptor expression. Enzyme-linked irnmunosorbent assay was used to quantify GABA-A receptor expression. RESULTS： Propofol, at a concentration of 0.5 and 2.5 μmol/L, increased proliferation of cultured rat hippocampal precursor cells inhibited by corticosterone, while bicuculline antagonized the effects of propofol （P 〈 0.05 or P 〈 0.01 ）. Corticosterone （100μmol/L） decreased expression of GABA-A receptor in the hippocampal precursor cells （P〈 0.05）, and GABA-A receptor expression was upregulated when propofol （2.5μmol/L） was added to the culture medium （P〈 0.05）. CONCLUSION： Low concentrations of propofol increased expression of GABA-A receptor. These results suggest that GABA-A receptor is involved in increased proliferation of cortisone-inhibited rat hippocampal precursor cells in vitro.

Effects of LW-AFC on anxiety-like behavior and immune function in corticosterone-induced mice

OBJECTIVE Chronic stress is one of the important factors in the development of many mental and neurological diseases,and cause damage to the central nervous system,affect animal emotions and damage the immune function of the body.The purpose of this study was to investigate the effects of LW-AFC which extracting from traditional Chinese medicine prescription Liuwei Dihuang decoctionon the anxiety-like behaviorand immune dysfunction abnormalities caused by chronic stress,and whether immune intervention affect the action of LW-AFC.METHODS Male BALB/c mice were subcutaneously injected with corticosterone(25 mg·kg^-1)for 28 d to establish a chronic stress model.Cyclophos⁃phamide(Cy,80 mg·kg^-1)was injected continuously for the initial three days,followed by once a week,LW-AFC(1.6 g·kg^-1)was given continuously for 28 d.Then investigate the emotion changes by open field and elevated plus maze tests,and detected the lymphocyte proliferation,lymphocyte subsets in peripheral blood,microglia and astrocyte expression,and inflammatory cytokines in peripheral blood and brain tissue.RESULTS The mice showed obvious depressive-like behaviorafter 28 d of continuous corticosterone injection.LW-AFC could significantly improve the anxietybehavior induced by corticosterone injection,but LW-AFC could not improve the anxietybehavior of mice by Cy intervention.The expression of glial cells in hippocampus of corticosterone-induced mice showed an upward trend,and the activation of microglia and astrocytes have significantly increase in corticosterone and Cy injected mice.LW-AFC significantly decreased the activation of microglia and astrocytes in corticosterone-induced mice with Cy intervention.This suggested that LW-AFC can reduce the damage of stress on the immune function of central nervous system under immunosuppres⁃sive state.Furthermore,LW-AFC could significantly up-regulate the proliferation of splenic lymphocyte stimulated by LPS and ConA,up-regulate the proportion of CD3+CD8+cells,reduce the proportion of CD4+/CD8+cells,decrease the secretion of inflammatory factors IL-6 and MCP-1 in plasma,and increase the level of anti-inflammatory factor IL-10 in plasma of mice induced by chronic corticosteroneinjection.While LW-AFC could promote the inflammatory factors TNF-α and IL-6in plasma,inhibit the secretion of anti-inflammatory factor IL-10 and inflammatory cytokine MCP-1 in hippocampus of corticosterone-induced mice with Cy intervention.CONCLUSION LW-AFC can improve anxiety-likebehavior induced by chronic stress,the Cy intervention affects the alleviation of anxiety-like behavior by LW-AFC as well as the regulation of immune function.The regulation of immune function might be the main way for LW-AFC to improve the function of central nervous system.

Xiaoyaosan-containing serum inhibits corticosterone-induced apoptosis in PC12 cells by downregulating [Ca^(2+)]_i overload

in hippocampal neurons is an important contributor to depression. [Ca^2＋]i of hippocampal neurons may raise rapidly with increased corticosterone concentrations, leading to apoptosis. The anti-depressant effects of Xiaoyaosan （Free and Easy Wanderer＇s Powder） may inhibit apoptosis by down regulating the [Ca^2＋]i overload. PC12 cells were containing with corticosterone to simulate neuronal injury, dosed with Xiaoyaosan-containing serum, and the following parameters were measured： apoptosis,[Ca^2＋]i overload, and changes in mitochondrial membrane potential （△ψm） and bioenergetics. High-dose Xiaoyaosan-containing serum （the dose of intragastric administration was 2.633 g herb/mL） not only inhibited morphological injury and cell apoptosis, but also suppressed [Ca^2＋]i overload induced by corticosterone. Xiaoyaosan also blocked the decrease in mitochondrial membrane potential and adenosine triphosphate levels induced by corticosterone. Xiaoyaosan dose-dependently suppresses corticosterone-induced apoptosis, potentially via inhibiting [Ca^2＋]ioverload, and prevents impaired mitochondrial bioenergetics by maintaining mitochondrial membrane potential and adenosine triphosphate production, providing a putative mechanism for its antidepressant effects.

Experimental Study of Effect of Corticosterone on Primary Cultured Hippocampal Neurons and Their Ca^(2+) /CaMKⅡ Expression

Summary: To explore the effect of different concentrations of corticosterone (CORT) on primary cultured hippocampal neurons and their Ca~2+ /CaMKⅡ expression and possible mechanism, the changes of hippocampal neurons were observed in terms of morphology, activity of cells, cell death, concentrations of cytosolic free calcium, and the expression of CaMKⅡ by using MTT assay, flow cytometry, fluorescent labeling of Fura-2/AM and Western blotting after 10~-7 , 10~-6 and 10~-5 mol/L of CORT was added to culture medium, The evident effect of 10~-6 and 10~-5 mol/L of CORT on the morphology of hippocampal neuron was found. Compared with control neurons, the activity of the cells was markedly decreased and [Ca~2+ ]_i increased in the neurons treated with 10~-6 and 10~-5 mol/L of CORT, but no change was observed in the neuron treated with 10~-7 mol/L of CORT. The death was either by way of apoptosis or necrosis in the cells treated with 10~-6 and 10~-5 mol/L of CORT respectively. The correlation analysis showed that a reverse correlation existed between [Ca~2+ ]_i and the expression of CaMKⅡ. Either apoptosis or necrosis occurs in the hippocampal neurons treated with CORT. The increased hippocampal [Ca~2+ ]_i is both the result of CORT impairing the hippocampal neurons and the cause of the apoptosis of hippocampal neurons and the decreased CaMKⅡ expression.

Corticosterone induced D-serine release deficit play an important role in long-term potentiation impairment by corticosterone in perforant path-dentate gyrus pathway

OBJECTIVE Previous studies showed that over activation of NMDA receptors may be a crucial cause of long-term potentiation(LTP)and cognitive impairment induced by stress or corticosterone.However,other studies showed that the function of NMDA receptors is insufficient since the NMDA receptors co-agonist D-serine could improve stress-induced cognitive impairment.The purpose of this study is to clarify whether over activation of NMDA receptors or hypofunction of NMDA receptors is involved in hippocampal impairment of LTP by corticosterone and the underlying mechanisms.METHODS Cort was injected subcutaneously 1 h before the high-frequency stimulation(HFS)to induce LTP impairment.NMDA receptor antagonists and agonists were administrated by icv.RESULTS Hippocampal LTP and object location recognition memory were impaired in corticosterone-treated mice.Corticosterone increased the glutamate level in hippocampal tissues,neither NMDA receptors antagonist nor its subtype antagonists alleviated impairment of LTP,while enhancing the function of NMDA receptors by D-serine did alleviate impairment of LTP by corticosterone,suggesting that hypofunction of NMDA receptors might be one of the main reasons for impairment of LTP by corticosterone.Further results showed that the level of D-serine and its precursor L-serine did not change.D-serine release-related protein Na+-independent alanine-serine-cysteine transporter-1(ASC-1)in the cell membrane was decreased and increasing D-serine release by the selective activator of ASC-1 antiporter activity alleviated impairment of LTP by corticosterone.CONCLUSION Taken together,this study demonstrates that hypofunction of NMDA receptors may be involved in impairment of LTP by corticosterone and reduced D-serine release may be an important reason for its hypofunction,which is an important complement to existing mechanisms of corticosterone-induced LTP and cognitive impairment.

Rapid effects of corticosterone and bovine serum albumin－conjugated corticosterone on the release of vasopressin

The hypothalamic slices (containing paraventricular and supraoptic nuclei) of SD rats sectioned with vibratome were incubated in a microchamber and arginine vasopressin (AVP) released from the slices was measured by radioimmunoassay. The effects of corticosterone (B) and bovine serum albumin-conjugated corticosterone (B-BSA) on AVP release were investigated. The results are as follows: (1) B and B-BSA inhibited AVP release within 20 min both in a dose-dependent manner from 10-7-10-4mol/L. At each dose, however, the rapid inhibitory effect of B-BSA was somewhat weaker than that of B. (2) RU486 could partially block the rapid inhibitory effects of both B and B-BSA,although it couldn't by itself change AVP release. But the dose of RU486 blocking the effect of B-BSA was an order of magnitude higher than that blocking the effect of B. (3) With the elevation of Ca2+ in the incubation medium, the rapid inhibitory effects of B and B-BSA were enhanced, while in the absence of Ca2+ their rapid effects attenuated. In these conditions, however, the effect of B-BSA was also weaker than that of B. These results suggested that the rapid inhibitory effect of B-BSA on AVP release from the rat hypothalamic slices was weaker than that of B, and the rapid inhibitory effect of B might be a non-genomic rather than classical genomic one, in which B might change the Ca2+ influx.