Mechanisms Of HtrA2-XIAP Involved In Neuronal Apoptosis And Its Potential Significance

Background:Aging is the sum of all the changes that take place in the body with the passage of time which ultimately lead to brain dysfunction with a series of morphological and biochemical metabolic changes. It has been reported that there was a significant loss of nerve cells during aging, both in animals and human beings, and it has been showed that both the degeneration of brain during aging and the pathological brain injury have the same pathophysiological process.The never cell play a key role in cerebral function as a basic unit, which implicated that the never cell loss along with aging was one of the most important factors lead to cerebral dysfunction and higher susceptibility with external stimuli, such as accidents caused by trauma. Traumatic Brain Injury (TBI) is one of common nerve injuries, and has higher disability and death rates. Nerve cell death or delayed neufonal apoptosis caused by direct trauma may was involved in brain dysfunction. Therefore the injured after trauma have a lower learning, memory ability and eventually a bad quality of life.The main cause of organ cell loss is cell death. Two most common forms of cell death are necrosis and apoptosis. Many studies suggest that both apoptosis and necrosis are involved in the heart of aging mice. But as time goes, the apoptotic cell death increase, but not necrosis. So we get a conclusion that the apoptosis play the major role in aging never cell loss. Apoptosis is an active programmed cell death characterized by a series of stereotypic morphological and biochemical features. Therefore, it's easier to inhibit apoptosis than to prohibit necrosis. Under the physiological conditions, apoptosis is manipulated, by the balance between pro-apoptotic and anti-apoptotic proteins, and these proteins also are manipulated by the balance of some upstream regulator proteins. As a result of complicated signal transduction of apoptosis, many particular cell types (such as never celll) couldn't maintain the balance exactly over some special cell stress (such as aging and trauma). Therefore, to determine the crucial pairs of pro-/anti-apoptosis signals is the most significant steps to solve aging problem, cerebral dysfunction, and other degenerated cerebral disease.XIAP (X chromosome-linked Inhibitor of Apoptosis Protein, XIAP) is the best-characterized and most potent Caspase inhibitor. It plays a crucial role by binding with Caspase-3 and Caspase-9.However, when apoptosis stimuli, Smac/DIABLO which is called the second mitochondria-derived activator of Caspases (Smac) or direct inhibitor of apoptosis-binding protein with low PI (DIABLO) and HtrA2/Omi are released from the mitochondria. Binding Smac/DIABLO or. HtrA2/Omi to XIAP disrupts the binding of XIAP to caspases and relieves the inhibition of apoptosis.Omi/HtrA2 is a mitochondrial conserved serine protease, and also one of important elements in endogenous apoptosis pathways. Compared to Smac/DIABLO, AIF or cytochrome c, HtrA2/Omi is a nuclear encoded mitochondrial serine protease that not only interacts with XIAP by binding activity and promotes caspase-dependent apoptosis, but also cleavage XIAP by its serine protease activity and promotes caspase-independent apoptosis. Recently, we have found that Omi/HtrA2 was increased in the aging heart, and there are many studies showed that Omi/HtrA2 also was involved in the aging rat cerebral cortex dysfunction. However, XIAP, which is regard as an endogen nous mitochondria anti-apoptosis protein, whether is associated with these mechanism or not. If so, to investigate which other endogen nous proteins could affect XIAP.Is it Smac/DIABLO, or HtrA2/Omi, or both of them? Whether the influence can reveal the more nerve cell loss, especially the cerebral cortex cell apoptosis, and cerebral dysfunction of aging and whether the influence could enhance susceptibility to traumatic brain injury have not been previously demonstrated. Therefore, we do the research as follows.(1)To observe the level of apoptosis in the cerebral cortex of aging rats. (2) To observe the effect of Omi/HtrA2-XIAP mechanism on cerebral cortex apoptosis of aging rats.(3) To observe the effect of Omi/HtrA2-XIAP mechanism on cerebral cortex apoptosis after traumatic brain injury. Objective1.To observe the level and possible pathway of apoptosis in the cerebral cortex of aging rats.2.To observe the expression of XIAP,Omi/HtrA2, Smac/DIABLO in the cerebral cortex of aging rats, and illustrate their roles in apoptosis.Methods1.Twenty rats was randomly selected from both male Sprague-Dawley adult rats (4-6months) and Sprague-Dawley aging rats (22-24 months). Each group has ten rats, provided with animal company in Jianyang City of Sichuan.2.Cerebral cortex apoptosis was analyzed by TUNEL (Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling) staining and Caspase-3 activity assay;3.The protein expression level of XIAP,Omi/HtrA2 and Smac/DIABLO was determined by Western Blot;4. The mRNA expression level of XIAP,Omi/HtrA2 and Smac/DIABLO was determined by Real time PCR.Results1. The level of apoptosis of cerebral cortex in both young and aging rats1.1 TUNEL stainingCerebral cortex apoptosis of young and aging groups was determined by highly sensitive TUNEL staining. The results showed that in the cerebral cortex of young rat group, there were much fewer positive staining nuclei, and the positive staining of apoptotic nuclei was increased significantly in cerebral cortex of aging rats.By calculating, compared with the young group(3.8±0.8),the positive staining apoptotic cells was significantly increased in aging group(11.4±0.6, P<0.05).1.2 Caspase-3 activity assay Caspase-3 is the pivotal protein in the final pathway of cell apoptosis. Theresults showed that compared to the young group(0.018±0.006), the Caspase-3 activity in the cerebral cortex was significantly increased in aging group(0.043±0.012, P<0.05)2.The Caspase-8,9,12 activities of cerebral cortex in both young and aging rats2.1 Caspase-8 activity assayCaspase-8 activity could quantitatively reflect the apoptosis activation by extrinsic pathway. The results showed that there was no statistics difference of Caspase-8 activity between the young(0.0046±0.0011)and aging group (0.0051±0.0006) (P>0.05), which suggested that the activation of apoptosis in cerebral cortex of aging rats was not through extrinsic pathway.2.2 Caspase-9 activity assayCaspase-9 activity could quantitatively reflect the apoptosis activation by intrinsic pathway. The results showed that compared with young group(0.0057±0.0009), the Caspase-9 activity was significantly increased in aging group(0.0069±0.0004, P<0.05), which suggested that the activation of apoptosis in cerebral cortex of aging rats may be through intrinsic pathway.2.2 Caspase-12 activity assayCaspase-12 activity could quantitatively reflect the apoptosis activation by endoplasmic reticulum stress pathway. The results showed that there was no statistics difference between the young(0.0023±0.0004)and aging (0.0028±0.0006) groups(P>0.05),which suggested that the activation of apoptosis in cerebral cortex of aging rats was not through endoplasmic reticulum stress pathway. 3.The protein expression of XIAP,Omi/HtrA2 and Smac/DIABLO in the cerebral cortex in both young and aging rats3.1 The protein expression of XIAP in the cerebral cortex in both young and aging ratsThe protein content of XIAP in the cerebral cortex was measured by western-blot. Compared with the young rats (0.048±0.005), XIAP protein expression in the cerebral cortex were significantly decreased in aging cerebral cortex(0.037±0.003,P<0.05).3.2 The protein expression of Omi/HtrA2 in the cerebral cortex in both young and aging ratsThe protein content of Omi/HtrA2 in the cerebral cortex was measured by western-blot. Compared with the young rats (0.064±0.008), Omi/HtrA2 protein expression in the cerebral cortex were significantly increased in aging cerebral cortex(0.092±0.011.P<0.01).3.3 The protein expression of Smac/DIABLO in the cerebral cortex in both young and aging ratsThe protein content of Smac/DIABLO in the cerebral cortex was measured by western-blot. There was no significant difference between the young and aging cerebral cortex. (P>0.05).4. The mRNA level of XIAP, Omi/HtrA2 and Smac/DIABLO in the cerebral cortex in both young and aging rats4.1 The mRNA level of XIAP in the cerebral cortex in both young and aging ratsThe mRNA content of XIAP was measured by Real-Time PCR. Compared with the young rats, the level of XIAP mRNA were significantly decreased in aging cerebral cortex (0.573±0.044, P<0.01).4.2 The mRNA level of Omi/HtrA2 in the cerebral cortex in both young and aging ratsThe mRNA content of Omi/HtrA2 was measured by Real-Time PCR. Compared with the young rats, the level of Omi/HtrA2 mRNA were significantly increased in aging cerebral cortex (1.782±0.036, P<0.01) 4.3 The mRNA level of Smac/DIABLO in the cerebral cortex between adult and aging ratsThe mRNA content of Smac/DIABLO was measured by Real-Time PCR. There was no significant difference of Smac/DIABLO in cerebral cortex between the young and aging groups.(P>0.05,see Figure 13)Summary1.The never cell apoptosis has been increased in aging rats, which was one of the important reasons of brain dysfunction.2.The main pathway of apoptosis in cerebral cortex was mitochondrial pathway, which results in apoptosis though Caspase-9 activation.3.Increasing Omi/HtrA2 and decreasing XIAP in cerebral cortex of aging rat suggested that more HtrA2/Omi induced XIAP degradation, caspases activation, and subsequently apoptosis in aging heart; Objective1.To observe the level of apoptosis after traumatic brain injury.2.To observe the effect of Omi/HtrA2-XIAP mechanisms on cerebral cortex apoptosis in traumatic brain injuryMethods1.Twenty rats was randomly selected from both male Sprague-Dawley adult rats (4-6months) and Sprague-Dawley aging rats (22-24 months). Each group has ten rats, provided with animal company in Jianyang City of Sichuan.2.The rat model of free-fall Brain injury model and the sham group. The improved method of Feeney's free-fall technology was used to built the free-fall brain injury rat model. Impact cone tip diameter of 4mm, height 2.5mm. Anesthetized rats, will be fixed in stereotaxic apparatus, prepared skin left after the cut along the midline scalp, separating periosteum, adjacent to the midline with a dental drill 2mm, close to the coronal suture after the opening round of a diameter of 5mm window, the dura mater intact. Use injuries impact damage to 600g.cm medium intensity. Pseudo-trauma group, only the scalp and skull cut open the window, not to impose against. Killed 4 hours after trauma.Young male SD rats (4-6months) were selected and aging SD rats (22-24 months old) were randomly divided into 2 groups:①Adult Sham group (n=6)②Adult Traumatic Brain Injury group (n=6)3.Cerebral cortex apoptosis was analyzed by TUNEL (Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling) staining and Caspase-3 activity, assay;4.The protein expression level of XIAP,Omi/HtrA2 and Smac/DIABLO was determined by Western Blot..Results1.The Caspase-3 activity in cerebral cortex after traumatic brain injury.Caspase-3 is the pivotal protein in the final pathway of cell apoptosis. Compared to the Sham group (0.019±0.008), the Caspase-3 activity in the cerebral cortex was significantly increased in traumatic brain injury group (0.034±0.006,P<0.05)2.XIAP, Omi/HtrA2,and Smac/DIABLO protein expression in the cerebral cortex after traumatic brain injury2.1 XIAP expression in cerebral cortex after traumatic brain injuryThe protein content of XIAP in the cerebral cortex was measured by western-blot. Compared with the sham group(1.063±0.013), Omi/HtrA2 protein expression in the cerebral cortex Were significantly decreased after traumatic bring injury (0.059±0.018, P<0.05).2.2 The protein expression of Omi/HtrA2 expression in cerebral cortex after traumatic brain injuryThe protein content of Omi/HtrA2 in the cerebral cortex was measured by western-blot. Compared with the sham group(0.103±0.024), Omi/HtrA2 protein expression in the cerebral cortex were significantly increased after traumatic bring injury (0.192±0.027, P<0.05).2.3 The protein expression of Smac/DLABLO expression in cerebral cortex after traumatic brain injuryThe protein content of Smac/DIABLO in the cerebral cortex was measured by western-blot. There was no significant difference of Smac/DIABLO mRNA in cerebral cortex between the sham and traumatic brain injury group (P> 0.05). Summary1.The cerebral cortex apoptosis was induced by traumatic brain injury, which 'may be one of the important mechanisms for cerebral dysfunction after traumatic brain injury;2.The mechanism of XIAP-Omi/HtrA2 was also involved in the cerebral cortex apoptosis after traumatic brain injury;Conclusion1.Apoptosis was increased significantly in cerebral cortex through the aging process of rats, and the apoptosis was mainly through the mitochondria pathway;2.Omi/HtrA2-XIAP mechanism was involved in cerebral cortex apoptosis, and increasing Omi/HtrA2 induced XIAP degradation,caspases activation, and subsequently apoptosis in aging heart;3.Omi/HtrA2-XIAP mechanism was involved in cerebral cortex apoptosis, and increasing Omi/HtrA2 induced XIAP degradation, caspases activation, and subsequently apoptosis in aging heart, which provided an important molecular target to ameliorate the. pathological damage induced by traumatic brain injury...