| Objective:β-caryophyllene, also called beta clove oil, is a compound of bicyclic sesquiterpanes. Its natural product can be extracted from clove leaf oil, clove oil, cinnamon leaf oil and so on. It exhibits strong anti-inflammatory and Anti-anxiety, antidepressant and analgesic properties in the current domestic and international study. But the research on the protective effects of β-caryophyllene on cerebral ischemia- reperfusion injury is less. This subject is designed to evaluate the effects of β-caryophyllene on nerve behavior, cerebral infarction volume, pathologic morphology, blood brain barrier and the expressions of COX-2、PPARγand NF-κB p65 of on the model of cerebral ischemia and reperfusion injury in rats in order to research the effects of β-caryophyllene on cerebral ischemia-reperfusion injury and possible mechanism.Methods:One hundred and thirty-eight male SD rats, weighing 250~280g, were randomly divided into six groups: sham operation group, model group, β-caryophyllene groups(34, 102, 306 mg × kg1-)and positive group(nimodipine 4 mg × kg1-).The model of cerebral ischemia-reperfusion injury( I/R) in rats was established by using the endoluminal thread.1. After 1.5 h of cerebral ischemia and 24 h of reperfusion,referenced the method of Zea Longa 5 points as the neurological deficit scores in rats; The infarction volumes were measured with 2,3,5-triphenyl- tetrazolium chloride(TTC) staining. The effect of β-caryophyllene on pathologic morphology of rats on the model of cerebral ischemia and reperfusion injury was observed by HE staining.2. The effect of β-caryophyllene on the water content of brain was measured by dry-wet weight method. The content of evens blue(EB) was measured to assess the permeability of BBB. The method of MPO was used to detect the effect of β-caryophyllene on the MPO activity of ischemic core cortex.3.The expression of AQP4,COX-2, PPARγ and NF-κB p65 in the ischemic core cortex were observed by immunohistochemisty staining.The contents of TNF-α,IL-6,IL-1β and PGE2 in the ischemic core cortex were measured by ELISA kits.4. The changes of claudin-5 and occluding and the expressions of claudin-5 and occluding protein were determined by Western bolt.Results:1. After 24 h of reperfusion,compared with the model group,β-caryophyllene improved the neural defective symptoms, reduced the neurological scores, reduced the infarction volume, improved the damage of neurons in the ischemic core cortex to varying degrees(P < 0.05, P < 0.01).2. After 24 h of reperfusion,compared with the sham operation group, the water content of brain in the model group increased significantly(P<0.01). Compared with the model group, the water content of brain in the middle and high doses of β-caryophyllene groups reduced(P<0.05). The content of evans blue in the model group showed a significant rise(P<0.01). Compared with the model group, the each dose of β-caryophyllene groups reduced the content of evans blue in different degrees. After 24 h of reperfusion,compared with the sham operation group, MPO activity of ischemic core cortex in the model group decreased to some extent, but the MPO activity in the middle and high doses of β-caryophyllene groups increased obviously.3.Immunohistochemical analysis showed that there were very little positive expression of AQP4, COX-2, NF-κB p65 and PPARγ in the cortex region of sham operation group. The cortex region of model group appeared a lot of AQP- 4, COX-2 and NF-κB p65 positive cells and a slight increase in the expression of PPARγ. Compared with the model group, the expression of AQP-4,COX-2 and NF-κ Bp65 in the cortex region of each dose of β-caryophyllene groups decreased significantly(P<0.05 or P<0.01), and the level of PPARγ protein in the cortex region of middle and high doses of β-caryophyllene groups significantly increased(P<0.01).ELISA results showed that the content of TNF-α 、IL-6 、IL-1β and PGE2 in the cortex region of the model group elevated, compared with the sham operation group, and the content of TNF-α 、IL-6 、IL-1β and PGE2 were significant down regulated in the middle and high doses of β-caryophyllene groups(P<0.01).4.The results of western blot showed that the expressions of claudin-5 and occluding decreased significantly(P < 0.01), the cortex region of model group appeared very little positive expression of Iκ Bα and PPARγ, compared with the sham operation group. And the levels of claudin-5, occluding, Iκ Bα and PPARγ protein in the cortex region of middle and high doses of β-caryophyllene groups increased significantly(P<0.01), compared with the model group.Conclusion:β-caryophyllene is beneficial to protect brain against cerebral ischemia reperfusion in rats. And its mechanism might be reduce the expression of AQP4, suppress the expression of inflammatory factor and adjust the signaling pathway of COX-2/ PPARγ / NF-κB p65, up-regulate claudin-5 and occluding protein expression to reduce the blood brain barrier damage. |
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