| Objective: The molecular technique was used to study changes of complex activities and related signal expressions of the respiratory chain in the hippocampus of the spleen qi deficiency and the interfering action of Sijunzi pill, revealing the possible central mechanism of spleen governing the transportation and transformation and providing the evidence for the biological mechanism of the treatment principle of nourishing qi to invigorate spleen.Materials and methods:1.The establishment and evaluation of spleen qi deficiency in rats: 60 male SD rats of SPF grade weighing 220 ± 10 g were randomly divided into the control, spleen qi deficiency(model group) and Sijunzi decoction pill(treating group), each 20 rats. The spleen qi deficiency of improper diet and fatigue was established, which was judged by the standard,including:(1) emaciation: the significant decrease of the body mass;(2) the poor appetite: the significant reduction of food and water intake, detected in metabolic cages;(3) mental fatigue: the significant reduction of the motion distance and vertical times in the open field during 5 min;(4) lack of strength: the significant decrease of the forelimb strength.2. The application of Sijunzi pill: Sijunzi pill was grinded into power and prepared with distilled water to the concentration of 2 g/m L, which was given to rats in the treating group by intragastric administration(1ml/100g), while distilled water was given to rats in the model group as the same way. Such activities lasted 2 weeks.3. The small intestinal propelling rate(SIPR): rats fasted without water deprivation for 24 h were intragastric administration(1 ml/100g) of 10% carbon powder suspension(5% active carbon and 10% acacia gum), then anesthetized with 10% chloral hydrate(0.35 ml/100g) after30 min. The SI from the pylorus to the ileocecal junction was carefully taken out, and SIPR was calculated as the following equation:(spreading distance of carbon power/the SI length)× 100%.4. preparation of hippocampal CA3 tissues: hippocampal tissues were taken out form anesthetized rats and hippocampal CA3 was separated under the microscope to be prepared in related ways for later tests.. Expressions of GLUT3 and its m RNA were tested with Western-Blot and RT-PCR,hippocampal concentrations of VIP, c AMP and AMPK were detected by ELISA,chromatometry was used to valuate activities of hippocampal mitochondrial complex, real time PCR was employed to detect PKA expression, Western-Blot was use to test ATP synthase expression, and immuohistochemstry was used to measure VIP receptor expression.6 Statistical Analysis The data were expressed as mean ± S.D., and statistical analysis was performed by SPSS 13.0 software. A value of p < 0.05 or less was considered to show statistical significance..Results:1. Pharmacological Effects of Sijunzi pill: compared with those in the control group, body mass, food intake, forelimb strength, standing times and SIPR were markedly reduced and the central residence time was significantly prolonged in the model group, which were corrected in the treating group.2. Effect of Sijunzi pill on the morphology of hippocampal CA3 neurons: neuronal mitochondria in the control group exhibited circular or oval shape and neuronal membrane was smooth, neuronal mitochondria in the model group were swelling and vacuolization,neuronal mitochondria showed normal shape.3. Effects of Sijunzi pill on the VIP concentration and expression in hippocampal CA3 tissue of model rats: compared with those in the control group, the VIP concentration and expression were decreased significantly, which were converted in the treating group.4. Effect of Sijunzi pill on the expression of GLUT3 in the hippocampal CA3 neurons of the model group: compared with those in the control group, GLUT3 and its m RNA expressions were reduced markedly, which were increased in the treating group.5. Effects of Sijunzi pill on the respiratory chain complex of hippocampal CA3 neurons:compared with those in the control group, activities of complex I, III and IV were reduced significantly, which were increased in the treating group obviously.6. Effect of Sijunzi pill on the expression of ATP synthase in the hippocampal CA3 neurons:compared with that in the control group, the ATP synthase expression was downregulatedsignificantly, which was upregulated in the treating group obviously.7. Effects of Sijunzi pill on the pathway of c AMP-PKA-AMPK in the hippocampal CA3neurons: compared with those in the control group, the c AMP concentration, expression of PKA m RNA were reduced significantly, and the AMPK concentration was increased markedly, which were all corrected in the treating group significantly.Conclusions:1.Model rats showed less standing times, prolonged central residence time, reduced movement distance and velocity and forelimb strength, which were converted by Sijunzi pill.2.The mitochondria of hippocampal neurons in the model group exhibited swelling and vacuolization, which were converted by Sijunzi pill.3.Hippocampal VIP concentration was increased and its receptor expression was upregulated in the model group, which were reduced and downregulated by Sijunzi pill.4.The expression of the membrane GLUT3 of hippocampal neurons in the model group was downregulated, which was upregulated by Sijunzi pill.5. Hippocampal neurons of model rats exhibited the reduction in the activities of the mitochondrial complex I, III and IV and expression of ATP synthase, which were converted by Sijunzi decoction.6.Concentrations and expressions of signals in the c AMP-PKA-AMPK pathway were decreased or downregulated in the model group, which were converted by Sijunzi decoction. |
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