Actin cytoskeleton reorganization correlates with polarization of secretory vesicle and cell morphology in the degranulation of mast cell subtypes in human colon tissues.

Mast cells play a central role in the intestinal immune response. To investigate the relationship between degranulation, cell polarization and the reorganization of actin cytoskeleton of mast cells, we used fluorescence or gold labeling methods to identify different mast cell subtypes in human colon. The reorganization of filamentous actin was visualized and then the polarization of secretory vesicles, as well as cell surfaces, was analyzed by fluorescence microscopy and electron microscopy. Our results first showed a diversity of filamentous actin assembly or disassembly within the contacting cell membrane of different mast cell subtypes. The polarization and degranulation of secretory vesicles was not only accompanied with the assembly and disassembly of filamentous actin at the cell periphery, but also with changes of cell surface polarization. Our study provides an insight into the local membranous structures and suggested correlations of cytoskeleton arrangement with the polarization of secretory vesicles and cell surface configuration during mast cell degranulation.

Short hairpin RNA mediated glypican-3 silencing inhibits hepatoma cell invasiveness and disrupts molecular pathways of angiogenesis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct glypican-3 (GPC-3) short hairpin RNA (shRNA) and investigate the effects of GPC-3 transcription silencing on hepatoma cell invasion and angiogenesis mechanisms.</p><p><b>METHODS</b>GPC-3-specific shRNA and non-target control shRNA were constructed and transfected into the human hepatoma cell lines HepG2, MHCC-97H, and Huh7. shRNA-mediated silencing of GPC-3 expression was confirmed at the mRNA and protein levels by fluorescence quantitative reverse transcription (FQRT)-PCR and western blotting, respectively. The effect of silenced GPC-3 expression on cell proliferation was detected by EdU and sulforhodamine B assays, on migration by wound healing (scratch) assay, on invasion by transwell chamber assay, and on apoptosis by luminescence assay of caspase-3/7 activity. The effect of silenced GPC-3 expression on angiogenesis-related signaling factors was detected by FQRT-PCR (for the glioma-associated oncogene homolog-1 hedgehog signaling factor, GLI1, and the beta-catenin Wnt signaling factor, b-catenin), immunofluorescent staining (for the insulin-like growth factor-II, IGF-II), and ELISA (for the vascular endothelial growth factor, VEGF). Pairwise comparisons were made by the independent sample t-test, and multiple comparisons were made by one-way ANOVA.</p><p><b>RESULTS</b>In all cell lines, transfection with the GPC-3-specific shRNA significantly reduced GPC-3 mRNA levels (% reduction as compared to the non-target control shRNA: HepG2, 89.2+/-6.0%, t = -25.753, P less than 0.001; MHCC-97H, 75.3+/-4.9%, t = -26.487, P less than 0.001; Huh7, 73.6+/-4.6%, t = -27.607, P less than 0.001); the GPC-3 protein levels were similarly reduced. The GPC-3 shRNA-silenced cells showed significantly reduced proliferative, migratory and invasive capacities, as well as significantly increased apoptosis. The shRNA-mediated GPC-3 silencing was accompanied by significant down-regulation of b-catenin mRNA (HepG2, 46.9+/-0.6%; MHCC-97H, 67.5+/-2.7%; Huh7, 56.3+/-8.4%) and significant up-regulation of GLI1 mRNA (HepG2, 49.2+/-28.6%; MHCC-97H, 54.6+/-24.4%; Huh7, 31.6+/-15.7%). At 72 h after transfection, the HepG2 cells showed significant down-regulation of VEGF protein (54.3+/-1.5%, t = 46.746, P less than 0.001).</p><p><b>CONCLUSION</b>GPC-3 contributes to migration, invasion, angiogenesis, and apoptosis of hepatoma cells, possibly through its interactions with the Wnt/b-catenin and Hedgehog signaling pathways. GPC-3 may represent a useful target for gene silencing by molecular-based therapies to treat hepatocellular carcinoma.</p>

Effects of N-n-butyl haloperidol iodide on the rat myocardial sarcoplasmic reticulum Ca(2+)-ATPase during ischemia/reperfusion.

We have previously shown that N-n-butyl haloperidol iodide (F(2)), a newly synthesized compound, reduces ischemia/reperfusion (I/R) injury by preventing intracellular Ca(2+) overload through inhibiting L-type calcium channels and outward current of Na(+)/Ca(2+) exchanger. This study was to investigate the effects of F(2) on activity and protein expression of the rat myocardial sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) during I/R to discover other molecular mechanisms by which F(2) maintains intracellular Ca(2+) homeostasis. In an in vivo rat model of myocardial I/R achieved by occluding coronary artery for 30-60 min followed by 0-120 min reperfusion, treatment with F(2) (0.25, 0.5, 1, 2 and 4 mg/kg, respectively) dose-dependently inhibited the I/R-induced decrease in SERCA activity. However, neither different durations of I/R nor different doses of F(2) altered the expression levels of myocardial SERCA2a protein. These results indicate that F(2) exerts cardioprotective effects against I/R injury by inhibiting I/R-mediated decrease in SERCA activity by a mechanism independent of SERCA2a protein levels modulation.

Correlation between epigenetic alterations in the insulin growth factor-II gene and hepatocellular carcinoma / 中华肝脏病杂志

To investigate whether epigenetic alterations in the insulin-like growth factor-II (IGF-II) gene that cause differential transcription or expression are correlated with onset and severity of hepatocellular carcinoma (HCC). Patient-matched specimens of HCC, paracancerous, and non-cancerous tissues were collected from 40 primary liver cancer patients. Epigenetic alterations in the promoter (P3) sequence of the IGF-II gene were analyzed by methylation-specific PCR (MSP) and IGF-II transcription was measured by RT-PCR. IGF-II protein expression and clinicopathological features were assessed by immunohistochemistry and microscopic observation. The rate of IGF-II P3 methylation was significantly lower in HCC tissues (0%) than in paracancerous tissues (vs. 47.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 100%; x2 = 80.000, P less than 0.001). IGF-II mRNA expression was significantly higher in HCC tissues (100%) than in paracancerous tissues (vs. 52.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 80.000, P less than 0.001). IGF-II protein expression was significantly higher in HCC tissues (82.5%) than in paracancerous tissues (vs. 45.0%; x2 = 12.170, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 56.170, P less than 0.001). IGF-II overexpression in HCC was significantly associated with degree of differentiation, extent of infiltrated serosa, size of tumor, and HBV-positive infection status. Epigenetic alterations in the IGF-II gene regulate its transcription and expression and are closely associated with HCC development and progression.

Expression features of glypican-3 and its diagnostic and differential values in hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the expression features of glypican-3 (GPC-3) and its diagnostic and differential values in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Rat hepatoma models were made and the dynamic expression features of GPC-3 protein and its gene were investigated by Western blotting and RT-PCR respectively. Liver specimens from 36 HCC patients were collected by self-control method and the expression and clinicopathological features of GPC-3 were analyzed by immunohistochemistry. Serum GPC-3 levels were quantitatively detected by ELISA and its efficiency for HCC diagnosis was evaluated in patients with liver diseases.</p><p><b>RESULTS</b>The incidence of GPC-3 was 0% in control, 83.3% in degeneration, 100% in precancerosis and 100% in canceration during dynamic formation of rat hepatoma, respectively. The positive GPC-3 was brown granule- like staining localized in membrane and cytoplasm in human HCC.</p><p><b>CONCLUSIONS</b>The GPC-3 positive rates were 80.6% in HCC, 41.7% in surrounding tissues and none in distal tissues (P < 0.01), respectively. No positive relationship presented between GPC-3 and differentiation grade or the number of tumor except of tumor size (Z = 2.941, P < 0.01). The incidence of serum GPC-3 was 52.8% in HCC patients except of one patient with cirrhosis. No significant differences were found between GPC-3 and sex, age, AFP, tumor number, Child classification or extrahepatic metastasis except of tumor size (χ² = 6.318, P < 0.05) and HBV infection (χ² = 23.362, P < 0.01). Combined detection of GPC-3 and AFP could rise up diagnosis of HCC. GPC-3 expression closely associated with HCC and might be useful for early diagnosis of HCC.</p>

Inhibitory effect of miRNA silencing hypoxia-inducible factor alpha subunit gene on the proliferation of HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of miRNA silencing HIF-1α gene on the proliferation of HepG2 cells.</p><p><b>METHODS</b>The eukaryotic expression plasmids of HIF-1α miRNA and report gene containing hypoxia-reponse element were constructed and transfected into HepG2 cells. The expressions of HIF-1α gene and protein were determined by real time-PCR and Western blotting. The expressions of HIF-1α, vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) were quantitatively detected by ELISA. The alterations of cell cycles and apoptosis rate were quantitatively measured by flow cytometry and Annexin V-FITC/PI double dyeing assay.</p><p><b>RESULTS</b>72 h after transfection the down regulations of HIF-1α mRNA and protein were 87% and 56% respectively, and the decrease of target gene was 46% in the report gene, 54% in VEGF and 36% in Ang-2, respectively. The apoptotic ratio of HepG2 cells was 22.46+/-0.61% (P < 0.01). The cell cycle changed greatly at the ratio of G1 (61.49+/-1.12%) and S (22.40+/-0.58%, P < 0.01). After being combined with doxorubicin, the apoptotic ratio increased to 36.99+/-0.88% and the ratios of G1 and S phases were upregulated to 65.68+/-0.91% and 19.47+/-1.34% respectively.</p><p><b>CONCLUSIONS</b>HIF-1α miRNA or / and doxorubicin can regulate the growth cycles of HepG2 cells, promote the cell apoptosis and inhibit the cell proliferation.</p>

[Determination of serum copper and zinc in different chemical forms by graphite furnace atomic absorption spectrometry with ethanol-EDTA precipitation method].

A simple and reliable method was developed for the determination of serum copper and zinc in different chemical forms by graphite furnace atomic absorption spectrometry (GFAAS) with ethanol-EDTA precipitation. The serum and ethanol were mixed with volume ratio 1 : 2. The mixture was incubated at 70 degrees C and ultra-centrifuged to precipitate proteins. Zinc and copper can be released from albumin after EDTA treating. Thus, macroglobulin-zinc, ceruloplasmin-copper, and albumin-bound zinc or copper could be determined by two proposed precipitation steps. The determination limit of copper (3sigma) was 1.2 microg x L(-1) and the recovery was 92.3%-104%, while the determination limit of zinc (3sigma) was 0.098 microg x L(-1), and the recovery was 90%-107%. This two-step precipitation method can be used to determine serum zinc and copper in various chemical forms in tumor, Wilson patients and heathy human.

N-n-Butyl haloperidol iodide protects against hypoxia/reoxygenation-induced cardiomyocyte injury by modulating protein kinase C activity.

N-n-Butyl haloperidol iodide (F2), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. We tested whether the myocardial protection of F2 on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by modulating protein kinase C (PKC) activity in primary cultured cardiomyocytes. Primary cultures of ventricular cardiomyocytes underwent 2-h hypoxia and 30-min reoxygenation. Total PKC activity was measured, and the translocation pattern of PKCalpha, betaII, delta and epsilon isoforms was assessed by fractionated western blot analysis. We investigated the association of PKC isoform translocation and H/R-induced injury in the presence and absence of the specific inhibitors and activator. Measurements included cell damage evaluated by creatine kinase (CK) release, and apoptosis measured by annexin V-FITC assay. In primary cultured cardiomyocytes exposed to H/R, PKCalpha, delta and epsilon were translocated, with no change in PKCbetaII activity. Total PKC activity, CK release and apoptosis were increased after H/R. Treatment with the conventional PKC inhibitor Go6976 reduced early growth response-1 (Egr-1) protein expression and attenuated apoptosis. The PKCepsilon inhibitor peptide epsilonV1-2 increased H/R injury without influencing Egr-1 expression. Pretreatment with F2 inhibited translocation of PKCalpha, increased translocation of PKCepsilon, and relieved the CK release and apoptosis. The protection of F2 was blocked in part by the conventional PKC activator thymeleatoxin (TXA) and epsilonV1-2 peptide. F2 significantly alleviated H/R-induced injury, which might be attributed to the combined benefits of inhibiting PKCalpha and activating PKCepsilon.

Effect of siRNA-mediated inhibition of nuclear transcription factor-kappa B on apoptosis of hepatocarcinoma cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of siRNA-mediated inhibition of NF-κB on apoptosis of hepatocarcinoma cells.</p><p><b>METHODS</b>Specific small interfering RNA Targeting NF-κB gene was synthesized and transfected into HepG2 cells by liposomes. Nested RT-PCR and quantitative RT-PCR were used to detect the mRNA expression of NF-κB. Immunohistochemistry, enzyme-linked immunosorbent assay and Western blot were performed to examine the protein expression of NF-κB. Annexin V-FITC was used to test cell apoptosis.</p><p><b>RESULTS</b>The expression of NF-κB in HepG2 cells (1.13+/-0.03) was significantly higher (t=27.02, P<0.05) than that in normal hepatocytes (0.29+/-0.07). The down-regulation of NF-κB expression was depended on the dosage of siRNA and the time after transfection. 72 h after siRNA transfection, NF-κB expression reduced by 93% and 62% at the mRNA and protein levels, respectively. The apoptosis of HepG2 cells increased by 85% with NF-κB inhibition.</p><p><b>CONCLUSIONS</b>NF-κB is abnormally active in HepG2 cells and NF-κB inhibition mediated by siRNA promotes HepG2 cells apoptosis. It suggested that NF-κB could be a potential target for HCC prevention gene therapy.</p>

Expression characteristics of hypoxia inducible factor-1a and its clinical values in hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic expression of hypoxia inducible factor-1alpha (HIF-1alpha) and its clinical values in hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>The dynamic changes of liver pathology, HIF-1alpha transcription and expression were observed through the hepatoma model. The self-control specimens from 35 human HCC patients were collected and the expression, cellular distribution, and clinicopathological features of HIF-1alpha and its gene was analyzed by immunohistochemistry, western blotting and nested- PCR, respectively.</p><p><b>RESULTS</b>Both levels of hepatic HIF-1alpha and HIF-1alpha mRNA expression increased during the HCC development course. The incidence of HIF-1alpha and the ratio of HIF-1alpha to beta-actin was 0% and 0.16+/-0.02 in the control rats, 77.8% and 0.29+/-0.04 in the denatured rats, 88.9% and 0.52+/-0.03 in the precancerous rats, and 100% and 0.84+/-0.02 in the cancerous rats respectively, with significant difference between the control group and any of the experimental groups (P = 0.000). The positive HIF-1alpha was brown and granule-like and mainly presented in cytoplasm and few in nucleus. The incidence of HIF-1alpha was 80% (28/35) in HCC and 100% (35/35) in its surrounding tissues. The clinical pathological features indicated HIF-1alpha expression associated with tumor size and differentiation degree the of HCC. No correlation was found between HIF-1alpha and tumor numbers or positive-HBsAg.</p><p><b>CONCLUSIONS</b>HIF-1alpha expression is associated with occurrence and development of HCC, and is perhaps a target molecule for HCC therapy.</p>

[Determination of trace free copper and zinc in serum samples by graphite furnace atomic absorption spectrometry after two-step precipitation of protein].

A method was developed for the determination of trace free copper and zinc in serum sample by GFAAS after two-step precipitation. The serum and ethanol were mixed with volume ratio of 1 : 2. The mixture was subsequently denatured at 70 degrees C and centrifuged to precipitate proteins. The determination, limit of copper (3sigma) was 1.2 microg x L(-1) and the recovery was 92.3% - 104%, and the determination. Limit of zinc(3sigma) was 0.098 microg x L(-1) and the recovery was 90%-107%. This two-step precipitation method can be used to determine non-protein-bound copper in tumor patients and healthy people.

[Optical and scanning electron microscopic observation of a novel small-caliber vascular graft in canine models].

OBJECTIVE: To observe the histopathological changes of a novel small-caliber vascular graft after implantation in canine theca interna under scanning electron microscope. METHOD: A 3 cm segment of the vascular graft (diameter of 4 mm) was implanted in an end-to-end fashion to bridge the severed carotid artery in 19 healthy dogs. Color Doppler sonography was performed 2 weeks after the operation to observe the patency rate of artificial blood vessel. At 1, 8, 12 and 24 week postimplantation, the arteries (4, 4, 6 and 5, respectively) were collected for optical and scanning electron microscopies after angiography to observe the patency of the arteries. RESULTS: Of the total of 19 arteries, occlusion occurred in 1 at 12 weeks and 1 at 24 weeks. Optical and electron microscopies showed that 1 week after implantation, slight fibroplasias and formation of red thrombus could be seen at the vascular anastomosis without endothelial cell lining. At 8 weeks, the host tissue grew into the lumen of the graft through the pores to form uniform neointima consisting of plenty of collagen fibers, but still without endothelial cells. At 12 weeks, discontinuous endothelial cells were seen to grow on the surface of the neointima. In the middle segment of the vascular graft, immature endothelial cells were found to grow in clusters. The structure of the neointima was loose in comparison with that at the anastomosis, with occasional inflammation cells. Twenty-four weeks after grafting, endothelial cells grew over the entire inner wall of the patent graft, and the surface of the neointima at the anastomosis was lined with continuous endothelial cells. CONCLUSION: The vascular graft can be useful for reconstruction of canine carotid artery defect and achieves good endothelialization 24 weeks after implantation.

An evaluation of transforming growth factor-beta 1 in diagnosing hepatocellular carcinoma and metastasis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic expressions of TGF-beta 1 and TGF-beta 1 mRNA at different stages of hepatocellular carcinoma (HCC) development and their use in clinical diagnosis.</p><p><b>METHODS</b>Hepatoma models were developed with 2-FAA using male Sprague-Dawley (SD) rats. Morphological changes of the rat liver histological preparations (H and E stained) were studied. The fragment of TGF-beta 1 gene obtained was amplified by nested RT-PCR. Dynamic change of TGF-beta 1 level was quantitatively analyzed by ELISA. The distribution of TGF-beta 1 in the cells and its gene expression were detected in human HCC tissues.</p><p><b>RESULTS</b>The progressive increases of hepatic TGF-beta 1 and TGF-beta 1 mRNA were observed in rat hepatocytes which progressed from granular degeneration, atypical hyperplasia and finally to HCC development induced by 2-FAA. The expression levels in HCC tissues were significantly higher than those in the normal and degenerative ones. TGF-beta 1 was shown in rat hepatocytes by immunohistochemistry. Plasma TGF-beta 1 was detected in 89.5% of all the patients with HCC, but it was detected in 93.3% of them who had an AFP less than 400 microg/L. TGF-beta 1 mRNA showed a stronger expression in HCC tissues. TGF-beta 1 mRNA was found in peripheral blood mononuclear cells from all HCC patients with extrahepatic metastasis.</p><p><b>CONCLUSION</b>TGF-beta 1 may participate in hepatocyte canceration. The overexpression of TGF-beta 1 and TGF-beta 1 mRNA could be useful markers for early diagnosis and predicting prognosis of HCC.</p>

Effects of N-n-butyl haloperidol iodide on myocardial ischemia/reperfusion injury and Egr-1 expression in rat.

We have previously shown that N-n-butyl haloperidol iodide (F2) derived from haloperidol reduces ischemia/reperfusion-induced myocardial injury by blocking intracellular Ca2+ overload. This study tested the hypothesis that cardio-protection with F2 is associated with an attenuation in the expression of early growth response gene 1 (Egr-1). In an in vivo rat model of 60 min coronary occlusion followed by 180 min of reperfusion, treatment with F2 significantly reduced myocardial injury evidenced by the reduction in release of plasma creatine kinase, myocardial creatine kinase isoenzyme and lactate dehydrogenase. In cultured neonatal rat cardiomyocytes of hypoxia for 3 h and reoxygenation for 1 h, F2 treatment attenuated necrotic and apoptotic cell death, as demonstrated by electron microscopy. Concomitant with cardio-protection by F2, the increased expression levels of Egr-1 mRNA and protein were significantly reduced in myocardial tissue and cultured cardiomyocytes as detected by reverse transcription-polymerase chain reaction, immunohistochemistry and immunocytochemistry. In conclusion, these results suggest that the protective effect of F2 on ischemia/reperfusion- or hypoxia/reoxygenation-induced myocardial injury might be partly mediated by downregulating Egr-1 expression.

Clinical values of vascular endothelial growth factor expression and microvascular density analysis in liver cancer specimens / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the roles of vascular endothelial growth factor (VEGF) in microvessel angiogenesis, development and metastasis of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>The cellular distributions of VEGF expression and microvascular density (MVD) in 36 HCCs were investigated, and the levels of total RNA and VEGF were detected in HCCs, Para cancerous, and distal cancerous tissues, respectively.</p><p><b>RESULTS</b>The incidence of VEGF was 63.9% in 36 cases of HCCs, 78.3% in non-encapsulated HCCs, and 90.9% in HCCs with extrahepatic metastasis, respectively. The VEGF expression was tightly correlated with MVD (t=4.49, P<0.01). No significant difference was found between VEGF or MVD and tumor diameter or differentiation degree. The level of total RNA in HCCs was lower but the VEGF level significantly higher than those of Para cancerous or distal cancerous ones (q=6.10, P<0.01).</p><p><b>CONCLUSION</b>The present data suggest that VEGF over expression and MVD abnormality are useful markers for vascular invasion and metastasis of liver tumors.</p>

[Effect of local testicular heating on spermatogenic cell apoptosis in rat].

OBJECTIVES: To study the effect of testicular local heating on spermatogenic cell apoptosis in rat. METHODS: Seventy male SD rats were divided into heat treatment group (43 degrees C) and control group (22 degrees C). Each group was further divided into seven sub-groups respectively according to the time of 12 hours and 1 days, 3 days, 6 days, 10 days, 50 days and 80 days after testicular local treatment. The spermatogenic cell apoptosis in all sub-groups was examined by means of electron microscopy, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUDP-nick end labeling(TUNEL) method. RESULTS: In the groups of heat treatment, spermatogenic cell apoptosis was detected by electron microscopy; flow cytometry showed that the percentage of cells with sub-haploid increased(P < 0.01); the percentage of positive TUNEL cells in the heat treatment groups was higher than that in the control group(P < 0.01). Initiation of spermatogenic cell apoptosis after testicular heating was not random but was highly selective. CONCLUSIONS: Local testicular heating could increase the spermatogenic cell apoptosis. The most sensitive cell is spermatocyte. Spermatid and sperm also display apparent changes. Heating can increase the apoptosis of spermatogonia in a long period.

Effects of phenytoin on morphology and structure of hippocampal CA3 pyramidal neurons of rats in chronic stress.

AIM: To investigate the effects of phenytoin (DPH) on morphological and structural changes of pyramidal neurons in hippocampal CA3 of rats induced by chronic stress. METHODS: Using Nissl staining, Golgi staining, and electron microscope, the morphology and structure of pyramidal neurons in hippocampal CA3 of rats were observed. RESULTS: Chronic stress resulted in loss of hippocampal CA3 pyramidal neuron from 39+/-4 to 35+/-4, shortening of total length of apical dendrite (from 196 microm+/-35 microm to 156 microm+/-33 microm, P<0.05), and ultrastructural degenerations of neurons. DPH markedly inhibited the decreases in number of hippocampal CA3 pyramidal neuron (38.4+/-2.2) and total length of apical dendrite (198 microm+/-36 microm, P<0.05), meanwhile, improved neuron ultrastructural degenerations caused by chronic stress. CONCLUSION: Chronic stress does damage to hippocampal CA3 pyramidal neurons and DPH protects hippocampus from damage induced by chronic stress.