Effects Of The Naoshenkang Capsule On Memory And Expression Of HIF-1α In Hippocampus Tissue Of Diabetes Mellitus Rats

Objective:To study the effect of the Naoshenkang capsule on memory and expression of hypoxia inducible factor-lalpha (HIF-la) in hippocampus tissue of diabetes mellitus rats.Methods1.Preparation of the diabetes mellitus rats model36heathy male Wistar rats were randomly devided into the normal control group (NC). the diabetes mellitus group (DM) and the Naoshenkang group (NSK). The DM and the NSK groups were feed with high cholesterol food for2weeks. All rats were fasted for16h, and then the DM and the NSK groups rats were given an intraperitoneal injection of60mg/kg streptozotocin. Then, after72h, a blood sample was drawn from vena caudalis, and rats with a non-fasting glucose level>16.67mmol/L were successful. The NC group was injected with same dose of buffer, and other operations were as same as the other groups. The NSK group was administered the Naoshenkang capsule once daily. The NC and the DM groups were given an equal volume of normal saline. All operations continued for12weeks.2.Gnenral condition observationWe observed the general condition of all rats, including the amount of food and water they took in everyday, the urine volume, the mental state and so on. The changes of weight were recorded every4weeks.S.BAEP test When rats were in narcosis, we recorded the changes of peak latency (PL) and interpeak latency (IPL) by using hypodermic needles. The recording electrode was put at the half-point of ears, while reference and earth electrodes were placed in the rear of mastoid ipsilateral and the tail separately. The sound stimulation of80Hz was given monaurally and repeated for1000times. The tests of two ears were operated separately every4weeks, and the Ⅱ, Ⅴ PL and Ⅲ-Ⅴ IPL were recorded in ms.4. Morris water maze experimentThe first part was making all rats to know where was the platform. First, we divided the maze pool into four quadrants with different makers in it. Then we chose one quadrant to set the platform. Second, all rats were put into the pool at an fixed point, and the time they swimming was limited in60s, if they cannot found platform in rule time, the operator would lead them to it. All rats were given15s to strengthen the memory of their spatial location. This part was operated in5days,4times a day. The second part was testing their memory ability. We removed the platform in the sixth day, all rats were put into the pool in training position, then we recorded the time and error times they cost to find the position of previous platform. This experiment also operated every4weeks, the date we collect was analysis by operator who did not participate in the test.5. Blood glucose levelAll rats were measured blood glucose through vena caudalis every4weeks.6. HE stainingAfter dewaxed, the slice was put into haematin for3min to dye nucleus, and then differentiated in the solution mixed with hydrochloric acid and alcohol for3s. Second, we used water to wash it for2min, then put it into tosin for2min to dye cytoplasm, followed by washing2min by water again. Last, we sealed the slice after it dehydrated by gradient alcohol, then we observed the shape of hippocampal neurons through the optical microscope.7. Immunohistochemical experimentAfter dewaxed, the slice was sealed by3%H2O2and5%serus separately after repairation. Second, we added the first antibody on it, then kept it in4℃until next day. Third, we added the second antibody on it, after which we used DAB to color the target hippocampal neurons. Last, we sealed the slice after it dehydrated by gradient alcohol, then we measured its IOD values by using the Kodak Image analysis system and Image-ProPlus software.8.RT-PCR textAccording to the methods recommended by Trizol nanual, we extracted the RNA from hippocampal tissue. RNA concentration is measured by spectrophotometer, the values of D260/280should be between1.8-2.0. Then we reversed RNA to cDNA, following the steps wrote in TaKaRa transcription kit. After that, we inserted the SYBR.(3-actin was considered as internal reference while HIF-la as purpose gene. Quantity one software was used to text the expression of HIF-la. Agarose gel electrophoresis was used to terrify the specificity of the PCR reacted products.Results:1. General stateIn the experiment, the rats of DM group took in more food and water. Besides, they grew slowly and their fur looked bleak. The weight of NSK group rats increased gradually, and the amount of food and water they took in are less than the DM group, while close to the normal rats. The NC group rats took in moderate diets, and their weight grew gradually day by day. At the same time, their active state is normal too.2. BAEPSince the4th week, the Ⅱ wave PL of DM group is longer than the NC and NSK groups (P<0.05or P<0.01). Since the4th week, the V wave PL of DM group is longer than the NC group (P<0.01). Since the8th week, the V wave PL of NSK group is shorter than the DM group (P<0.05or P<0.01). Since the4th week, the Ⅲ-Ⅴ IPL of DM group is longer than NC and NSK groups (P<0.05or P<0.01).3. Morris water mazeSince the4th week, the escape latency of DM group is longer than the NC and NSK groups (P<0.05or P<0.01).4. Blood glucose levelBlood glucose of rats in the DM and NSK groups are in high level throughout the experiment. In contrast, after the8th week, blood glucose levels of NSK group rats were obviously lower than DM group (P<0.05or P<0.01).5. HE stainingNormal hippocampal neurons arranged orderly, its shape looked like chiseled and clear with an integrated nuclear membrane. The hippocampal neurons of DM group had a chaos order, normal cells are mixed with heteroideus cells which appeared in the form of deeply dyed nucleus. The hippocampal neurons of the NSK group were significantly better than DM group, cells arranged more order, a spot of cells have deeply dyed nucleus, the number of normal cells close to the NC group.6. ImmunohistochemistryThe expression of HIF-la increased both in the DM and the NSK groups (P<0.01or P<0.05) at the4th week. Compared with the DM group, the NSK group had an obviously decrease in the expression of HIF-la since the8th week (P<0.01or P<0.05).7. RT-PCRThe mRNA expression of HIF-la increased both in the DM and the NSK groups (P<0.01) at the4th week. Compared with the DM group, the mRNA expression in the NSK group was obviously decreased since the8th week (P<0.01or P<0.05).Conclusion:The Naoshenkang capsule has a beneficial effect in decreasing blood glucose level, ameliorating the hypoxia of hippocampus, inhibiting the expression of HIF-1α and hippocampal neuronal apoptosis, and thus increasing memory function of diabetics.