The Effect Of Carvacrol On Hippocampal Neurons In Pilocarpine-induced Seizures And The Mechanism

Background and objectiveEpilepsy(EP) is a common diseases of nervous systerm. There are more than 9 million people with epilepsy in China, and the growth rate is 650-700 hunderd every year. Repeated sezuires carry burdens not only to patients and families, but also to the society, so it’s very important to find the pathogenesis.Endoplasmic reticulum is a very important organelle, and it’s mainly responsible for the transduction of cellular Ca2+ signal, involving in the steady state of Ca2+ and what is important is transporting the proteins after they were folded. Endoplasmic reticulum is extremely sensitive to stress and various states of stress could be induced by endoplasmic reticulum stress(ERS), such as high blood pressure, oxidative stress.Moderate endoplasmic reticulum stress could increase the expression of GRP78 and induced the unfolding protein reaction(URR) to reduce the accumulation of misfolded proteins to reduce the damage to the brain. But excessive endoplasmic reticulum stress would active the related apoptosis signaling pathways and trigger apoptosis. CHOP and caspase12 are ERS apoptosis- related protein.Carvocrol(CAR) is generally exists in all kinds of natural oil. Recent studies showed that CAR has many fuctions such as anti-tumor, antioxidant, anti apoptosis and anti-depresent. However, the role of CAR on status epilepticus rats is still unknown.In our study, we observed the behavior of rats after pilocarpine-induced SE and the hippocampus neurons damage and the effect of CAR on epileptic rats. In addition, we investigated the different doses of CAR on the expression of endoplasmic reticulum stress related molecular GRP78(glucose regulated protein-78), caspase 12 and CHOP(C/EBP homologous protein) and investigate the possible mechanism. Materials and methodsAll male SD rats are 6-8 weeks, and the weights are about 200-250 g. We randomized devided them into CON group, PILO, PILO+CAR30(30 mg/kg) and PILO+CAR60(60 mg/kg) four groups, PILO group were including 2 h, 8 h, 24 h, 72 h group. Each group contain 20 rats. Rats were intraperitoneal inject of lithium chloride(127mg/kg) previously, then twenty hours later they were given the intraperitoneal injection of pilocarpine(30mg/kg). 30 min befor the injection of pilocarpine, the scopolamine were intraperitoneal injected used to antagonize the peripheral chonlinegic response of pilocarpine. PILO+CAR30 group and the PILO+CAR60 need to intraperitoneal injection of CAR(30mg/kg, 60mg/kg) befor the injection of pilocarpine. Equal volume of normal saline instead of pilocarpine were intraperitoneal injected in CON group. When the status epilepticus lasts 60 min, we intraperitoneal injected the diazepam(10mg/kg) to terminate the pilocarpine induced sezuires. The Racine sezuires grading criteria were used as standard and we think the model is making success when sezuires up to Ⅳ/ grade. Half rats of the four groups Ⅴand the PILO 24 h were anesthesized after taking brain perfusion, using the Nissl and TUNEL staining to detect the hippocampal injury and apoptosis 24 h after SE. The remaining group were anesthesized 24 h after SE, the rats were anesthesized at the corresponding time in PILO group,then they were isolated the bilateral hippocampus. Western blot were used to detected the expression of the GRP78、caspase12 and CHOP. Results1.Behavioral observation : rats in con group don’t have any seizures. PILO group, PILO+CAR30 group and PILO+CAR60 group have seizures after the pilocarpine induced epilepsy and the seizure grade of them up to Ⅳ/ grⅤ ade.2.Nissl staining results: Compared with con group, PILO group, PILO+CAR30 group, PILO+CAR60 group appear obvious damage after the pilocarpine induced epilepsy and the number of hippocampal neurons significantly reduced(P<0.05). Compared with the PILO group, the number of hippocampal neurons increased in PILO+CAR30 group(P<0.05). Compared with PILO+CAR30 group, hippocampal neurons were significantly increased in PILO+CAR60 group.3.TUNEL staining results: Compared with CON group, PILO group, PILO+ CAR30 group and PILO+CAR60group, the number of apoptosis neurons significantly decreased(P<0.05). Compared with the PILO group, The numbers of apoptosis cells significantly increased(P<0.05). Compared with PILO+CAR30 group, numbers of apoptosis cells increased in PILO+CAR60 group(P<0.05).4.Western blot analysis: Compared with con group, the expression of GRP78 rise 2h after SE,and 8h reached the peak and continue to 24 hours in PILO group. Caspase12 and CHOP rise 2h after SE, 24 hours reached the peak and last 72 hours. Compared with PILO group, the expression of GRP78, caspase12 and CHOP increased in PILO group. The expression of GRP78 significantly increased and the expression of caspase12 and CHOP decreased in PILO+CAR group. Compared with PILO+CAR30 group, the expression of GRP78 significantly increased and the expression of caspase12 and CHOP decreased in PILO+CAR group(P<0.05). Conclusion1.GRP78,caspase12 and CHOP increased after SE, instructing that epilepsy induce the endoplasmic reticulum stress.2.Carvacrol has protective effect the hippocampal neurons in epileptic rats and tthe effects was dose-dependent. We thought the undelying mechanism were increasing the expression of GRP78, inhibiting the caspase12 and CHOP.