Inulicin Intervening AD Progress In Alzheimer's Disease Model Rats

Alzheimer's disease (AD) is an ageing-related neurodegenerative disorder with new cases every year (20-30 million individuals) worldwide. AD starts with mild memory deficits, gradually progresses to cognitive and behavioral spheres due to the extensive neuronal and synaptic loss, the intraneuronal formation of neurofibrillary tangles and the extracellar deposition of amyloid-β(Aβ) plaques in susceptible regions of the brain. Chronic inflammatory response to antigens such as Aβand NFT seem to have a role in AD process. Pro-inflammatory mediators induced by microglia and astroglia activation contributes to neuronal dysfunction and cell death, which draw patients to cognitive and behavioral dysfunction and dementia. These imply that inflammatory process inhibition may intervene AD development.The inulicin [containing three specific sesquiterpene lactones, britannilactone (BL), 1-O-acetylbritannilactone (1-O-ABL), and 1,6-O,O-diacetylbritannilactone (1,6-O2-ABL)], isolated from inula britannica-L, is used in inflammation diseases treatment by the traditional Chinese herbal medicine. In this study, we treat AD model rats with inulicin to compare its effect on inflammatory cytokines expression so as to AD progress.RESULTS1 Inulicin inhibiting COX-2 and iNOS expression in AD rats ippocampus Sprague-Dawley (SD) rats weighing of 364.5±15.0 g were microinjected with Aβ25-35 into the hippocampus to induce AD model rats.In inulicin treatment group, the animal were pretreated with inulicin (contain ABL 2.66mg /ml) dissolved in tween-80 3 days before operation, then inulicin was administered to rats at dose of ABL 26mg/kg/d by gastric gavage orally for 18 days (n=4). An equal volume of tween-80 (3 ml/rat/d) was administrated orally to model group rats (n=4). The control group animals were treated by the same operation as the other two groups but with microinjection of NS (n=4). At 21 days after surgery, the rats were killed by blood-bleeding, and the hippocampus were quickly isolated for histological and Western blot analysis.1.1 Effect of inulicin on COX-2 expressionWe studied COX-2 Expression in AD rats and matched controls. COX-2 expression were more intensive in hippocampus cell of model rats than that of control rats. Western blot analysis showed the same results as Imunohistochemistry staining. Our data demonstrated that Inulicin can inhibit COX-2 expression.1.2 Effect of inulicin on iNOS expressionNO is a gaseous free radical, which is generated by enzymes of the nitric oxide synthase family. Excessive NO induce the cell death. In order to evaluate the anti-inflammatory activity of inulicin, we examined the iNOS expression in AD rats and matched controls. iNOS expression were more intensive in hippocampus cell of model rats than that of control rats. Western blot analysis showed the same results as Imunohistochemistry staining. Our data demonstrated that Inulicin can inhibit iNOS expression.1.3 Innulicin effect on histomorphology of AD rats hippocampus.Hippocampus sections of the three groups were compared by H&E-staining . There were a large of neurons in control group rats, disposed in regularity multiplayer. The nuclei were large, round and clear, so do the nucleoli. The neurofibra were close-up in regularity. Typical AD pathological changes were found in model rats. There were less neurons in indiscriminate disposition. The nuclei were pycnotic and hyperchromic, and displayed in polygonal or triangle. The neurofibra werere sparse. Microglia were recruited around neurons. These indicate that the model were successfully duplicated. Aforesaid pathological changes were ameliorated in inulicin-treated rats hippocampus.1.4 Effect of inulicin on learning and memory To assay behavioral defects, the three group rats were measured for escape distance. After training (day 5), all rats on day 6 were given three trials where they swam in the pool for 60 s with the platform removed. The results were significantly different for model rats vs. innulicin-treated rats, and so that were control rats in the day 2, 3 and 4(P<0.05).But there was no difference between innulicin-treated rats and control rats in the day 2, 3 and 4(P>0.05).Also there was a significant difference between model rats and innulicin-treated rats on the crossing of former platform location(P<0.05). Taken together, all these data indicated that innulicin administration attenuated dysfunction of learning and memory in AD rats.2 Effect of inulicin on the expression of NF-κB and PPARγin AD rats hippocampus2.1 Effect of inulicin on NF-κB activation and translocation NF-κB is a transcriptional factors control many inflammatory cytokines expression including COX-2 and iNOS. Therefore we investigated whether inulicin control COX-2 and iNOS expression through NF-κB pathway. The cytosolic and nuclear NF-κB p50 subunit expressional levels in rats hippocampus were detected by immunochemistry and Western blot. Aβinjection into hippocampus increased p50 translocation to the nuclei, and inulicin attenuated this response. The positive cells for NF-κB were positively stained in model rat hippocampus cell but not in that of control rat. Western blot analysis showed the same results as immunochemistry staining. Our data demonstrated that inulicin can strongly inhibit NF-κB activiton.2.2 Effect of inulicin on PPARγexpressionPPARγis related to inflammation and Aβdeposition in AD brain. We therefore examined the PPARγexpression in hippocampus after Aβinjection. The PPARγexpressional levels in rats hippocampus were detected by Western blot. Aβinjection into hippocampus decreased PPARγexpression, and inulicin attenuated this response. These data demonstrated that innulicin strongly increased PPARγexpression.3 Inulicin inhibiting cell apoptosis of AD hippocampus 3.1 Effect of inulicin on Bcl-2/Bax expressionBcl-2 is the prototypical antiapoptotic member of a large family of proteins involved in the modulation of cell death. Bax facilitates apoptosis by anti-Bcl-2. To assess the mechanism of inulicin inhibiting apoptosis of hippocampus neurons, Bcl-2 and Bax expressional levels were detected by Western blot and Iimmunohistochemical assays. Aβinjection into hippocampus increased Bax expression, and inulicin attenuated this response.The expression of Bcl-2 expression was no difference among the three group rats. These data demonstrated that innulicin could inhibit apoptosis through Bax down-regulation.3.2 Effect of inulicin on casepase-3 expressionCasepase-3 is the executor of apoptosis process and activated casepase-3 induces the cell death regardless of any apoptosis channels. Therefore we studied Casepase-3 expression in AD rats and matched controls. Casepase-3 expression were more intensive in hippocampus cell of model rats than that of control rats. The positive cells for casepase-3 were positively stained in model rat hippocampus cell but not in that of control rat. Western blot analysis showed the same results as Imunohistochemistry staining. Our data demonstrated that Inulicin can inhibit Casepase-3 expression.3.3 Apoptosis ratio detection of hippocampus neurons by flow cytometryApoptosis ratio of hippocampus neurons were detected by flow cytometry. The ratio were 3.55±0.22, 3.62±0.41 and 4.69±0.54 respectively. The results were different for model rats vs. innulicin-treated rats (P < 0.05). Our data indicated that innulicin inhibited the apoptosis by Aβinjection into hippocampus.Conclusions1 Inulicin inhibits inflammation in hippocampus by reducing the expression of iNOS and COX-2 in AD rats.2 Innulicin administration could attenuate learning and memory dsyfuction in AD rats by its anti-inflammatory effect. 3 Innulicin inhibits the expression of pro-inflammatory gene by down-regula- ting NF-κB expression and up-regulating PPARγexpression.4 Iinulicin could inhibit the apoptosis by Aβinjection into hippocampus by down-regulating Bax and casepase-3 expression, but not up-regulating Bcl-2 expression.