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Objective: To investigate the protective effects and mechanism of Zuogui Jiangtang Jieyu Formulation (ZGF) on hippocampal neuron of neurovascular unit (NVU) induced by autophagy in diabetes mellitus with depression (DD). Methods Hippocampal neurons, astrocytes and brain microvascular endothelial cells from SD rats were primitively isolated, purified and cultured, and then immunocytochemistry staining was employed to identify primitive cells. The DD model of NVU was established by applying intervention of high glucose (150 mmol/L) and cortisone (200 μmol/L) after co-culture system was built in vitro. The cultured cells were randomly divided into normal group, blank serum group, model group, and medicated serum of ZGF group. Intracellular calcium levels (Ca2+) on hippocampal neuron of NVU was detected by Furo-3/AM staining. The expression of autophagy marker Beclin-1, LC3-II, and GluR2/Ca2+/mTOR signaling pathway key proteins was detected by high content analysis. The apoptosis of hippocampal neurons was observed by Tunel staining. Results:Compared with the normal group, Ca2+ levels was remarkably increased, expression of autophagy marker Beclin-1 and LC3-II was dramatically up-regulated, proteins expression of GluR2 and mTOR was obviously down-regulated while AMPK up-regulated, and the apoptosis in hippocampal neuron of NVU was significantly increased in model and blank serum group. Compared with the model group, intracellular calcium levels was obviously decreased, expression of autophagy marker Beclin-1 and LC3-II was down-regulated, GluR2/Ca2+/mTOR signaling pathway proteins expression of GluR2, mTOR was up-regulated while AMPK down-regulated, furthermore the apoptosis of hippocampal neurons was significantly decreased in ZGF group. Conclusion: ZGF has significant protective effects on hippocampal neuron of NVU induced by autophagy in DD, and its mechanism is related to the GluR2/Ca2+/mTOR signaling pathway and apoptosis.
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Objective To observe the effects of Zuogui Jiangtang Jieyu Prescription (ZGJTJYP) on pathological morphology of hippocampal neurons in rats of diabetes mellitus with depression (DD); To discuss its protective mechanism of action. Methods DD rat model was established by tail vein injection of STZ combined with chronic stress. Totally seventy-two SPF rats were randomly divided into normal group, model group, positive medicine group, ZGJTJYP low-, medium-, and high-dose groups, with 12 cases in each group, and were given corresponding medicine. After the last administration, blood glucose and behavioral changes were measured in each group. HE staining, Nissl staining, and Golgi staining were used to observe the changes of pathological morphology in rat hippocampus. The expression of Caspase-3 in hippocampus was detected by immunohistochemistry. Results Compared with the normal group, blood glucose level was significantly increased in the model group; the number of neurons in the hippocampus decreased in the model group; the apoptosis increased in the model group; the neuronal soma showed deformation and reduced dendritic spines and other pathological damages in the model group. After intervention of ZGJTJYP, hyperglycemia and depression-like status of the model rats were relieved; the number of hippocampal neurons increased significantly; neuronal morphology basically returned to normal. The expression of Caspase-3 in ZGJTJYP high-and medium-dose groups was significantly lower than that in model group. Conclusion ZGJTJYP can significantly improved blood glucose and learning and memory abilities in DD rats, which may be related to slowing hippocampal injury and apoptosis, and restoring neuronal quantity and morphology.
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Objective To investigate the effects of S 100B, AQP4, and CX43 on the function of the blood brain barrier in the hippocampus of diabetic rats with depression .Methods Rats were divided into the diabetic group [ two weeks of high-fat diet and injection of Streptozotocin (STZ, 38 mg/kg)], depression group(chronic unpredictable stress for 28 days) , the diabetes mellitus with depression group ( combined with the above two methods ) , and the control group .The behavior of rats was evaluated with open-field test and Morris test .The expressions of AQP 4 and CX43 in rat hippocampus blood-brain barrier were detected by immunocytochemistry .Serum S100B level was detected by ELISA.Results Compared with control group, the number of autonomic activity and the space exploration times were decreased, the escape latencies were significantly longer in the Morris water maze test(P<0.05 or P<0.01) of the diabetes group and depression group; Serum S100B levels increased significantly ( P<0.01);the expressions of AQP4 and CX43 were decreased(P<0.01).Compared with diabetic group, the number of auto-nomic activity and the space exploration times were decreased , the escape latencies were significantly longer in the Morris water maze test( P<0.05 or P<0.01) of the diabetes group and depression group;Serum S 100B lev-els increased significantly( P<0.05);the expressions of AQP4 and CX43 were decreased(P<0.05 or P<0.01). Conclusions S100B, AQP4 and CX43 expression disorder may be one of the mechanisms of diabetes mellitus complicated with depression.
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Objective To investigate the effects of Zuogui Jiangtang Jieyu Prescription on neurotrophic effects of astrocytes in diabetes mellitus rats with depression. Methods The diabetes mellitus with depression rat models were established by composite method, and then were randomly divided into 5 groups: model group, positive group, Zuogui Jiangtang Jieyu Prescription high-, medium-, low-dose groups, with 12 rats in each group. 12 normal rats were set as control group. Each administration group was given relevenat medicine for gavage for continuous 4 weeks. Open-field test was used to evaluate the depression-like behavior. The expression of GFAP in astrocyte and MAP2 in neuron were tested by immunohistochemistry. The expression of protein and mRNA of BDNF, GDNF, and NGF were tested by Western blot and RT-PCR. Results Compared with the control group, the activities of rats in model group were significantly reduced. The expression of GFAP increased, while the expressions of MAP2, BDNF, GDNF and NGF decreased. Compared with the model group, the depression-like behavior of rats in model group were significantly improved. The expression of GFAP decreased, while the expressions of MAP2, BDNF, GDNF and NGF increased, and GFAP decrseaed significantly. Conclusion The secretion function of astrocyte can be improved by Zuogui Jiangtang Jieyu Prescription. Its anti-depression and neuron-protection function might be correlated with the enhancement of neurotrophic effects of astrocytes.
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Objective To investigate the regulating effects of Zuogui Jiangtang Jieyu Formula (ZJJF) on Bcl-2, Bax and Caspase-8 in hippocampus neuron damage in diabetes mellitus with depression (DD). Methods Hippocampal neurons from 18 d pregnant rats were primitively cultivated, and then combination of glucose and corticosterone was used to construct DD simulation environment. Cultivated hippocampal neurons were randomly divided into normal group, blank serum group, model group, positive medicine (metformin+fluoxetine) serum group and to-be-tested medicine (ZJJF) serum group. Normal group and model group were given same amount culture medium, while other group were given relevant amont of 10％ medicine serum or blank serum. After modeling intervention for 18 h,Hoechst staining was used to detect the apoptosis of hippocampal neurons. The expression of Bcl-2, Bax and Caspase-8 was detected by high content analysis. Results Compared with the control group, hippocampal neuron dendrites ruptured or decreased, neural network connection decreased, cells showed significant staining, broken, uneven distribution of light spots, the expression of Bcl-2 protein decreased significantly (P<0.05), but Bax and Caspase-8 were increased (P<0.05, P<0.01). Compared with the model group, hippocampal neurons in both positive medicine serum group and ZJJF serum group gradually recovered. Hoechst staining showed that the nuclei were significantly homogenized, local highlights were significantly reduced, Bcl-2 protein expression levels were significantly increased (P<0.05, P<0.01), while Bax and Caspase-8 were obviously down-regulated (P<0.05, P<0.01). Conclusion ZJJF has protective effects on hippocampal neurons in DD of model rats, and its mechanism is related to regulating the expression of Bcl-2, Bax and Caspase-8 in hippocampus neuron.
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Objective: To investigate the effects of Zuogui Jiangtang Jieyu Formula (ZJJF) on protein expression of CoIV, ZO-1, and α-SMA in hippocampal blood-brain barrier (BBB) in diabetes mellitus (DM) rats with depression. Methods: DM rat model with depression was established by the combination of high-fat diet, injection of Streptozotocin (STZ, 38 mg/kg), and chronic stress. The model rats were randomly divided into five groups, DM with depression group, metformin (0.18 g/kg) + fluoxetine (1.8 mg/kg) group, high-, mid-, and low-dose (32.82, 16.41, and 8.20 g/kg) ZJJF groups (n = 10). After 4 weeks of administration, the morphological changes of BBB were observed under the electron microscope, the expression of CoIV, ZO-1, and α-SMA in rat hippocampul BBB was detected by immunocytochemistry. Results: Compared with the control group, the expression of ZO-1 and α-SMA in the model group was decreased (P < 0.05), and the expression of CoIV was increased (P < 0.05). Compared with the model group, the expression of ZO-1 and α-SMA in metformin + fluoxetine group and high-dose ZJJF group was increased (P < 0.05, 0.01), and the expression of CoIV was decreased (P < 0.05). Conclusion: ZJJF could repair the damage of BBB in DM rats with depression, and the mechanism may be related to the expression regulation of CoIV, ZO-1, and α-SMA.
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Objective: To investigate the effects of Zuogui Jiangtang Jieyu Fang (ZJJF) on the blood glucose and blood lipids in diabetes mellitus (DM) rats with depression. Methods: DM rat model with depression was established by the combination of high-fat diet, injection of Streptozotocin (STZ, 38 mg/kg), and chronic stress. The model rats were randomly divided into five groups, the DM with depression group, metformin (0.18 g/kg) combined fluoxetine (1.8 mg/kg) group, high-, mid-, and low-dose (20.53, 10.26, and 5.13 g/kg) ZJJF groups. After 4 weeks of administration, blood glucose, HbA1c, lipids, and insulin resistance (IR) were assayed by biochemical techniques. The corticosterone (CORT) level in plasma was detected by ELISA method. HE staining was used to observe the adrenal pathological changes of MD rats. Results: Compared with the control group, the levels of blood glucose, HbA1c, total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL-C) were significantly higher, and the level of high-density lipoprotein (HDL-C) was significantly lower. The CORT level in plasma was significantly higher (P < 0.05). The significant pathological changes existed in adrenal gland were observed by HE staining. Compared with the model group, the blood glucose was significantly stable, IR, HbA1c, TC, TG, LDL-C, and CORT levels in plasma of rats in ZJJF group were significantly decreased, HDL-C level was significantly elevated, while the adrenal pathological changes were reduced. Conclusion: The metabolic disorders of blood glucose and lipids could be improved by ZJJF, which might be correlated to the down-regulation of the CORT level for protecting the adrenal gland.