作者：聂庆珠，沙倩，王英爽,归东梅，刘致力，高殿文
作者单位：110004 中国辽宁省沈阳市，中国医科大学附属盛京医院眼科
【摘要】目的:研究慢性高眼压过程中大鼠视网膜Caspase9表达的变化及应用AG对其表达的影响,探讨AG可能存在的对慢性高眼压视网膜的保护作用。
方法:应用免疫组化、RTPCR和Western blot的方法观察应用AG及未应用AG的慢性高眼压大鼠视网膜在慢性高眼压后不同时间点的形态学变化及Caspase9表达的变化。
结果:与正常对照组相比，随着高眼压时间的延长视网膜逐渐出现可察觉的形态学变化，于高眼压的第21d视网膜变薄，节细胞数量减少；在此过程中，Caspase9表达增多，与形态学变化相一致。而应用AG者其形态学变化和Caspase9表达变化较小。
结论:凋亡相关基因Caspase9在慢性高眼压视网膜损伤过程中发挥了作用，AG通过下调其表达对视网膜起保护作用。
【关键词】 视网膜；慢性高眼压；凋亡；氨基胍（AG）；半胱天冬酶9（Caspase9）
Expression of caspase9 affected by AG on retina of rats with chronic IOP elevation
QingZhu Nie, Qian Sha,YingShuang Wang, DongMei Gui, ZhiLi Liu, DianWen Gao
Department of Ophthalmology, Shengjing Hospital Affiliated to China Medical University, Shenyang 110004, Liaoning Province, China
Abstract
AIM: To study caspase9 expression on rat retina in the process of chronic elevation of IOP and the changes with the application of amino guanidine (AG), thus to investigate potential protective function of AG to rat retina with chronic elevation of IOP.
METHODS: Immunohistochemistry, RTPCR and Western blot were used to observe retinal morphology and expression of caspase9 at different time points of rat with chronic IOP elevation, both affected or not affected by the application of AG.
RESULTS: Compared with control group, as time passed retina of experimental group gradually had detectable morphological changes. On 21st day of chronic IOP elevation, retinas became thinner and the quantity of retinal ganglion cells (RGCs) decreased; caspase9 expression increased, consistent with the morphological changes.The group using AG presented relatively smaller morphology changes and less expression of caspase9.
CONCLUSION: Apoptosisrelated gene caspase9 played a part in the process of chronic IOP elevation; AG protects retina by downregulating expression of caspase9.
KEYWORDS: retina; chronic IOP elevation; apoptosis; amino guanidine; caspase9
INTRODUCTION
Intraocular hypertension is a major risk factor of glaucoma injury. Nowadays the treatment of glaucoma is mainly based on the decrease of IOP with medicine or operation. However, a simple decrease of IOP is not enough to prevent the progressive neural injuries caused by glaucoma[1]. Therefore, there might be also some other factors working. At present, glaucoma is considered as a multifactor controlling disease and the final common pathway is the apoptosis of RGCs, while the mechanisms of how all these factors induce apoptosis of RGCs and the regulations of apoptosis genes are still unclear. Therefore, it is very important to investigate how these apoptosisrelated genes are involved in the apoptosis of RGCs at different levels and how they are regulated, so that we can provide better treatment for patients. Caspase is a family of intracellular cysteine endopeptidases, which is the most important apoptosisrelated gene found by now[2]. It is classified into two groups based on their primary constructions and the size of Nterminal prodomain: (1) Promoter: caspase which triggers on DNA fragmentation during apoptosis. (2) Effector: caspase which induces apoptosis directly by enzymolysis to substrate protein. Caspase9 is a promoter. Amino guanidine (AG) is an inhibitor of induced type of nitricoxide synthase, which is safe and effective and extensively applied in the research of myocardial ischemia and cerebral anoxia but comparatively less used in glaucoma investigation. It is reported by Neufield and his coworkers that AG is an optic nerve protector of rat model of chronic IOP elevation, but the mechanisms are to be further investigated. This experiment is designed to explore the connection between the expression of caspase9 and RGCs in chronic IOP elevation, and to testify whether AG is involved in the regulation of caspase9 to play the role of neuroprotection. This experiment will be performed at levels of gene and protein. It will investigate apoptosisrelated genes and the changes of its protein expression in normal retinal tissue and retinal tissue of IOP elevated rats with and without AG application. We performed this experiment to reveal the mechanisms of optic nerve injury and protection for glaucoma to some degree, and we look forward to applying all our findings to clinical treatment. Hopefully we can find the best neuroprotectant in this multiple pathway system and provide a new idea for the treatment of glaucoma.
MATERIALS AND METHODS
Materials
Laboratory animals Fifty male Wistar rats, weighing between 200300g, were supplied by experiment animal department of China Medical University. The animals and experimental conditions followed laboratory animal regulations of State Science and Technology Commision. The animals were randomly divided into 3 groups, which are 10 in blank control group (20 eyes), 30 in chronic IOP elevated group (60 eyes), and 30 in chronic IOP elevated with AG application group (60 eyes).
Reagents of immunohistochemistry Protein antibody: rabbit antirat caspase9 (Wuhan Boster Biotechnology Co., Ltd), SABC kit (Wuhan Boster Biotechnology Co., Ltd) were pure.
Reagents of RTPCR MMLV reverse transcriptase (Promega, USA), Oligo (dT) (Promega, USA), Heat resistant Taq DNA polymerase (Takara Biotechnology (Dalian) Co., Ltd)，TaKaRa RNA PCR (Takara Biotechnology (Dalian) Co., Ltd), PCR primer: designed by primer Express 3.0 based on GeneBank.
Reagents of western blot N, N’methylene bisacrylamide (BBI, USA), N, N, N, NTEMED (Fluka, USA), sodium dodecylsulphate (SDS), DTT (Sigma, USA), NC filter (Amersham, UK), and Normal goat serum (Wuhan Boster Biotechnology Co., Ltd) were also provided.
Methods
Rat model of chronic IOP elevation The rats were anesthetized by intraperitoneal injection of 100g/L chloral hydrate 0.3mL for every 100g body weight; bulbar conjunctiva was cut and two superficial venous tributaries were burnt (signs of successful burn: episclera venous blood flow disappeared on the distal end of the burnt point; distension and darkness of the vessels near corneoscleral limbus); bulbar conjunctiva was reset with TobraDex drops and paste as eyedrop application. IOP was also measured with TONOPEN Ⅱ, and the measurement time points were before the operation, half an hour after the operation, the 7th day, the 14th day, the 21st day, and the 28th day. IOP that is 40% beyond preoperative value (916mmHg) means modeling was successful. To group with AG application, AG (Sigma, USA) was given 1 day ahead of modeling with 1g in 1L of their drinking water, but the total amount will be under 150mg/(kg·d). The modeling procedure was all the same.
Immunohistochemistry After sampling, fixation, dehydration and paraffin imbedding were performed according to the instruction of the kit. Positive cells were those with yellow or brownishyellow granules deposited in cytoplasm or nuclei. We selected 5 discontinued high power fields from each section to assess the expression intensity with etaMorph/BX51 microgram analytical system through data analysis of the determination of integrated OD of positive cells.
RTPCR Total RNA was extracted from retinal tissue; then reverse transcription product of 1 μg total RNA was amplified (reaction conditions: 95℃ 5min, 94℃ 30sec, 54℃ 30sec, 72℃ 40sec, total 35 cycles，72℃10min). Caspase9 primer (sense：5’ACC ACA GTC CAT GCC ATC AC3’；antisense: 5’TCC ACC ACC CCT GTT GCT GTA3’)，length of product was 452bp. Internal control GAPDH primer (sense：5’TTT AAG CTC TCA GAA GAC ATG 3；antisense：5’TGT TGA AGT ACA GAC AGT ACC CCC A3)，length of product was 363bp. Optical density of PCR product was detected by AGE and relative absorbance ratio was calculated to internal control.
Westernblot Retinal tissue was cut up and was added with 100mL cytolysis solution, cell protein was extracted to carry out SDSPAGE electrophoresis and then was transmembraned to nitrocellulose filter. Acted with first and second antibody of anticaspase9, labelled by horseradish peroxidase (HRP) and then DAB coloration was performed.
Statistical Analysis Analysis was done with SPSS13.0 and the result was presented with mean±standard deviation ( ±s). The comparison between medication group and nonmedication group was analyzed by students t test, while the comparison between model group and control group was analyzed by oneway ANOVA test.
RESULTS
Change of RGCs and Thickness of Nerve Fiber Layer in Rats with Chronic IOP Elevation On 21st day after modeling, the retina became thinner and the number of RGCs decreased; however, the change of medication group was relatively mild. Among three groups at least two of them had distinctive difference according to the analysis by Levene homogeneity test for variance with the result of P=0.137(>0.05), which means homogeneity of variance; intergroup multiple comparison test indicated that, in terms of thickness comparison, compared with control group the variances of other two groups both had statistical significance; thickness comparison between medication group and unmedication group had statistical significance(Table 1).
Distribution and Intensity of Expression of Caspase9 by Immunohistochemistry It is illustrated in figure 1. Control group: only trace quantity of positive expression was detected in layer of ganglion cells in the retina of normal rats. Immune positive cells had yellow brown nuclei or cytoplasm stained; Unmedication model group: expression increased at the 3rd day, and at the 7th and 14th day it was the highest. It was seen bodies of ganglion cells were big and round while bodies of microglia were long and thin. Besides ganglion cell layer, there was also positive expression in inner plexiform layer and inner nuclear layer. There was a small quantity of expression in ocular cones and rod cells. On the 28th day, a certain amount of nuclei were yellowbrown stained in cells mentioned above. Medication group: change of time course was similar to unmedication model group, but with less extent. IOD values which indicate expression intensity of caspase9 in rat retinas from model groups are shown in table 2.
Expression of Caspase9 mRNA Detected by RTPCR Expression of caspase9 mRNA was enhanced with the extension of time. The strongest expression was on 7th and 14th day, and expression decreased after that (Figure 2). Levene variance homogeneity test, P=0.179 (>0.05), showed homogeneity of variance. Oneway ANOVA analysis, P<0.05, showed among the expressions of caspase9 mRNA at different time points, there was at least one value significantly different from other time point values; time point multiple comparison (Turky method) test showed that except 3rd day IOP elevated group, values of each group had statistical difference compared with control group. Expressions of caspase9 mRNA of medication model group, compared with unmedication model group by t test, P<0.05, which suggest that it decreased significantly. Relative absorbance ratios of caspase9 mRNA expressions in rat retinas of various groups are shown in table 3.
Expression of Caspase9 Protein Detected by Western Blotting Expression of caspase9 protein was enhanced with the extension of time. The strongest expression was on 7th and 14th day, and decreased thereafter (Figure 3). Levene variance homogeneity test, P=0.153 (>0.05), which showed homogeneity of variance. Oneway ANOVA analysis, P<0.05, which showed among the expressions of caspase9 protein at different time points, there was at least one value significantly different from other time point values; time point multiple comparison (Turky method) test showed that except 3rd day IOP elevated group, values of each group had statistical differences compared with control group. Expression of caspase9 protein of medication model group, compared with unmedication model group by t test, P<0.05, which showed it decreased significantly. Relative absorbance ratios suggesting caspase9 protein expressions in rat retinas of various groups are shown in table 4.
Figure 1Intensity and distribution of caspase9 protein expression of control group and model groups （略）
A: control group; B: 7th day after IOP elevation; C: 14th day after IOP elevation; D: 28th day after IOP elevation; E: 7th day AG+IOP elevated; F: 14th day AG+IOP elevated; G: 28th day AG+IOP elevated
Table 1Thickness change of retina in rat of chronic IOP elevation with and without AG application（略）
Note:a, b indicate that the difference between the two groups P<0.05. a indicates statistical significance exists when we compared control group with other groups separately; b indicates statistical significance exists when we compared model group with AG application with other groups respectively
DISCUSSION
In China, most animal models for glaucoma research are ischemiareperfusion models, which have disadvantages for observation of retina protection, while reports about morphological changes under chronic IOP elevation are rare. This investigation approached the protection of AG to retina suffering from glaucoma through observing the expression of apoptosisrelated gene caspase9 and its change with application of AG, in which chronic IOP elevation modeling method was generally accepted overseas. Studies indicate that there could be multiple factors involved in the mechanism of retinal injury due to IOP elevation, but there is only one outcome, which is apoptosis of RGCs[1]. Some researchers[2,3] Figure 2Expression of caspase9 mRNA in retina of rats with chronic IOP elevation suggested that patients with glaucoma lost their sights though their photoreceptors were still functional because of chronic progressive death of RGCs. Apoptosis of RGCs in chronic IOP elevation aggravates with the extension of time, which indicates this modeling method can be used in the research on retina protection of chronic IOP elevation.
A: control group; B: 3rd day after IOP elevation; C:7th day after IOP elevatipn; D: 14th day after IOP elevation; E: 21st day after IOP elevation; F: 28th day after IOP elevation; G: 3rd day AG+IOP elevation; H: 7th day AG+IOP elevation; I: 14th day AG+IOP elevation; J: 21st day AG+IOP elevation; K: 28th day AG+IOP elevation
Figure 3Expression of caspase9 protein in retina of rat with chronic IOP elevation（略）
Table 2Expression intensity ( IOD ) of caspase9 in rat retina of each model group（略）
aP<0.05, bP<0.01
Table 3IDV ratio of expression of caspase9 mRNA in retina of rats from each group（略）
bP<0.01
Table 4IDV ratio of expression of caspase9 protein in retina of rats from each group（略）
aP<0.05, bP<0.01
Biological Characteristics of Caspase Family Caspase[4] is a family of intracellular cysteine endopeptidases. They play a key role in inflammation and mammalian apoptosis. Fourteen proteases of this kind have been identified till now, which are numbered Caspase 114 in the order they were found out. They are divided into two classes based on the lengths of their Nterminal prodomains. (1) Promotor caspase: have long prodomains and also named long prodomain caspase, including caspase1, 2, 4, 5, 8, 9 and 10, which trigger on DNA fragmentation during apoptosis. (2) Effector caspase: including caspase3, 6, 7, 14, have short prodomains, which induce apoptosis directly by enzymolysis to substrate protein. Caspase family extensively exists in all kinds of cell tissues of mammals, as well as in ocular region. Caspases can be detected in photoreceptor cells[5,6], RGCs[7] and so on, among which caspase3 is found out to have high degree of similarity with the product of cell death gene ced3, which is principal medium triggering RGCs death. When positioning active caspase3 on RGCs of rat whose axons had been cut off, it was found that its active fragment p20 coexisted with TUNEL and its proteoclastic activity increased[8]. Caspase9 is an important activator of caspase3, bax/bcl2 dependent cytochrome C released from mitochondrion to cytoplasm and combined with Caspase activator Apaf1 to induce formation of oligomerization complex, mobilize and activate precaspase9. Caspase9 is formed and activates caspase3 and initiates apoptosis in the end.
Influence of AG on Expression of Caspase According to results of immunohistochemical method in this investigation, there was only trace amount expression of caspase9 in normal rat retina, which was localized in ganglion cell layer. As time went by, positive reaction cells were found in inner plexiform layer, inner nuclear layer and ocular cones and rod cells, which demonstrated the expression of caspase9 was enhanced, and reached the peak on the 14th day, then decreased to a relatively stable level. Detections of caspase9 mRNA by RTPCR and caspase9 protein by Western blotting both shared similar change course with results of immunohistochemical method, which indicated that it was a continuous procedure of apoptosisrelated factor caspase9 from transcription to translation to protein. All the above manifested with the existence of chronic IOP elevation the expression of caspase9 increased and the injury to RGCs was continuous. Therefore, the retina protection to patients suffering from chronic IOP elevation should be performed in a long term. Besides, another phenomenon was observed: expression of caspase9 in ocular cones and rod cells during 7th 14th days of chronic IOP elevation, which gave us a better explanation of early phase decrease of color vision and contrast sensitivity in chronic IOP elevation.
With the application of AG, the degree of retina thinning diminished compared with unmedication model group (P<0.05), and the expression of caspase9 in retina of rat with chronic IOP elevation was significantly inhibited. Immunohistochemical method, RTPCT and Western blotting showed respectively that caspase9 IOD, mRNA and expression of protein were all inhibited and through statistical analysis the differences were significant (P<0.05). The results mentioned above indicated AG as a relatively specific inhibitor of iNOS, could inhibit directly or indirectly the expression of caspase9 to protect retina in chronic IOP elevation.
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