芥子气皮肤损伤毒理学及对抗药物研发策略应用基础研究
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摘要
芥子气(sulfur mustard, SM)素有“毒剂之王”之称,是糜烂性毒剂的典型代表。皮肤是芥子气的最主要中毒靶器官之一,芥子气染毒皮肤后能够引起红斑、水肿、起疱、糜烂、坏死并容易造成二次感染,伤口愈合缓慢。
自芥子气出现于第一次世界大战以来,针对芥子气防护以及损伤治疗的相关研究一直持续,并向深入发展。但是,迄今为止,芥子气毒理学机制仍未完全阐明,针对芥子气中毒的特效抗毒药物仍然属于空白,临床上仅能采取对症治疗措施,究其原因,可能与芥子气作为双功能烃化剂,其毒理学作用具有多水平、多靶点的特点有关。因此,芥子气皮肤损伤毒理学机制以及基于相关研究基础之上的特效抗毒药物研究策略的探索和有效实施,仍是国内外芥子气研究领域关注的工作重点之一。
基于课题组前期工作积累,并结合国外研究进展,本课题选取了与芥子气皮肤损伤具有密切关系的谷胱甘肽硫转移酶(glutathione-S-transferase, GST),以及组织因子途径抑制剂-2 (tissue factor pathway inhibitor-2, TFPI-2)作为研究重点,旨在进一步了解GST在芥子气诱导细胞凋亡中的作用,探索其能否成为缓解芥子气毒性的新途径,同时,利用生物工程技术体外制备TFPI-2,探讨其作为一种丝氨酸蛋白酶抑制剂,在芥子气损伤预防和治疗研究中的价值,并试图通过这种关注和积累芥子气皮肤损伤不同环节作用资料的研究,以逐步对特效抗毒药物研究和开发策略的制定提供有意义的帮助。
1.芥子气诱导GST活性变化规律与细胞凋亡情况的关系研究。
在芥子气皮肤损伤小鼠耳廓模型上,GST活性变化具有剂量依赖性和时间依赖性。随着芥子气染毒剂量加大和染毒时间延长,GST活性下降,至染毒剂量为160mg/ml时所有时间点,GST活性显著下降;所有剂量组染毒后72h,GST活性显著下降,与课题组前期蛋白质组学实验结果取得了较好的一致性,明显提示,芥子气染毒后,皮肤组织依赖于GST的对自由基及芥子气的清除能力有所减弱。
将芥子气作用于原代培养的乳鼠皮肤成纤维细胞,观察由其诱导细胞发生凋亡的情况,结果发现,在芥子气浓度为100μM染毒后16h,细胞凋亡明显,此时细胞内GST活性显著下降,至芥子气染毒后24h,死亡细胞显著增多。结果提示芥子气诱导的细胞凋亡与GST活性降低在某种程度上可能具有关联性。
2.增加细胞内GST对芥子气诱导细胞凋亡效应的影响
从GST含量丰富的小鼠肝脏提取总RNA,经过PCR扩增反应,测序并与NCBI序列库比对,证实取得正确的GST基因,将该基因与pCMV真核表达载体进行载体构建,为后续细胞转染奠定基础。
通过脂质体转染技术,将携带有克隆基因片段GST的真核表达载体转染至HaCaT细胞系,并利用免疫印迹对其表达进行验证,将该细胞称为HaCaT/GST。芥子气染毒两种细胞(染毒浓度为100μM,检测时间为16h),HaCaT/GST与HaCaT细胞相比,细胞凋亡百分比明显下降,证实了单独转染GST对于芥子气诱导的细胞凋亡有防护和对抗作用。
在上述染毒条件下,分别于染毒后6h、12h、24h,检测HaCaT细胞内GSH、SOD和MDA变化情况。结果表明,随着芥子气染毒时间延长,GSH含量降低,脂质过氧化反应产物MDA含量逐渐增加,SOD被芥子气刺激产生的自由基所耗竭,含量逐渐下降,细胞内氧化还原平衡被破坏,最终引起细胞损伤。提示芥子气诱导的细胞凋亡与氧化还原系统失衡有关,并且GSH耗竭在该过程中可能起一定作用。
3.TFPI-2分子克隆及其第一结构域的基因克隆、蛋白表达与纯化为了探讨TFPI-2能否缓解芥子气毒性,从TFPI-2含量较高的人胎盘调取该基因,并从其全长基因中将含有Kunitz结构域1(first Kunitz-type domain, KD1),即TFPI-2/KD1基因片段进行扩增。分别将TFPI-2和TFPI-2/KD1构建至原核表达载体,利用原核表达的方法将两种蛋白进行体外表达,结果表明TFPI-2以包涵体形式表达,而TFPI-2/KD1以可溶的形式表达于细胞内上清液,提示TFPI-2/KD1可能具有生物活性,不需经过复性过程。因此,在后续的实验研究中,我们对TFPI-2/KD1进行了纯化,进一步探讨其活性和功能。
4.TFPI-2/KD1的功能研究
4.1 TFPI-2/KD1的活性验证
采用明胶酶谱法检测TFPI-2/KD1能否抑制H1299癌细胞株分泌的基质金属蛋白酶;采用发色底物法检测TFPI-2/KD1能否抑制胰蛋白酶。结果表明,TFPI-2 /KD1浓度为20μM时能够显著的抑制MMP-2、MMP-9;在一定浓度范围内,随TFPI-2/KD1浓度的升高,对胰蛋白酶的抑制作用呈现增强的趋势。提示TFPI-2 /KD1具有抑制丝氨酸蛋白酶的活性。
4.2 TFPI-2/KD1对于芥子气诱导炎症因子分泌的影响
采用放射免疫分析法检测炎症因子IL-6、IL-8和TNF-α。与正常细胞相比较,100μM芥子气染毒HaCaT细胞后8h,上清液中IL-6、IL-8和TNF-α均显著分泌。TFPI-2/KD1蛋白为1mg/ml以及2mg/ml时,能够显著性抑制芥子气诱导IL-8分泌;TFPI-2/KD1蛋白浓度为1mg/ml时,能够显著性抑制芥子气诱导IL-6分泌;而各浓度组TFPI-2/KD1(0.5mg/ml、1mg/ml以及2mg/ml)对于芥子气诱导的TNF-α分泌升高却没有抑制作用。基于以上结果,我们推测TFPI-2/KD1通过影响细胞因子分泌,可能具有对抗芥子气诱导炎症反应的作用。
4.3芥子气染毒家兔皮肤血流变化与病理损伤研究
利用激光多普勒仪检测不同剂量芥子气(1μl、2.5μl、5μl、10μl)染毒家兔背部皮肤血流变化情况,连续观察至染毒后第三天,将动物处死,做石蜡切片检测皮肤的病理损伤程度。
结果显示,芥子气染毒后,染毒皮肤短时间内即出现血流加快,提示染毒部位可能出现急性炎症反应,血管扩张;随着时间的延长,中心区域供血减弱,出现环形供血区域,提示染毒部位的血管可能产生损伤,周围组织代偿性供血。随着染毒时间延长和剂量增加,环形供血区域更加明显。
病理切片结果显示,家兔正常背部皮肤可见表皮、真皮、附属器及肌肉组织;1μl芥子气染毒后表皮出现糜烂、溃疡、表皮下大疱(>5mm)、毛囊和皮脂腺有瘤样增生;随着染毒剂量加大,部分表皮消失,表皮可见坏死及渗出物;真皮层血管扩张、充血、水肿,有大量的炎细胞浸润并深达附属器;急性炎症反应明显(中性粒细胞为主);染毒剂量为10μl时,表皮出现灶性坏死,出血明显。
4.4 TFPI-2/KD1干预芥子气引起皮肤炎症的初步探讨
TFPI-2/KD1(给药剂量为1mg/ml,4次/日)对伴随芥子气急性损伤时所观察到的,体现急性炎症性反应特点的血液流变规律没有明显影响;病理结果显示,与对照组比较,TFPI-2/KD1给药后染毒皮肤炎症反应有所减轻,表现为中性粒细胞浸润数量减少,表皮灶性坏死程度减轻。但总体上没能非常有效地改善芥子气染毒所致皮肤病理损伤,芥子气染毒皮肤表面出血、灶性的炎症反应等病理变化仍十分常见。提示TFPI-2/KD1对芥子气所致皮肤损伤治疗效果有限,而这一现象在芥子气的治疗中具有普遍提示性:单一药物干预效果不理想。
简而言之,在芥子气皮肤损伤过程中GST以及TFPI-2通过不同作用环节起到相应的作用,前者主要与芥子气诱导的细胞凋亡相关,而后者可能通过对丝氨酸蛋白酶的抑制效应,部分地对抗了芥子气诱导的急性炎症反应。但是,单一途径的干预效果仍然十分有限,体现了芥子气损伤的多部位、多靶点特征,提示针对芥子气损伤特效抗毒药物研究和开发策略不应仅局限于单一环节,而应该努力寻找最为关键的环节和靶点,并尽量兼顾其多部位、多靶点特征。
Sulfur mustard (SM),“the king of the chemical warfare agents,”is a typical vesicant agent. Skin is the major target for SM., the most prominent toxic effects of which are extremely slow healing lesions and blisters resulting in ulcer and vesicle of the skin, and promoting secondary infections.
Although the toxicity of SM has been investigated since it was used in the World War I. Presently, the mechanisms underlying sulfur mustard-induced skin damage are not fully elucidated and no antidote exists, apart from its characteristics of multiple stratification and multiple targets. Current treatment strategy is designed to relieve symptoms. Critical questions are proposed to increase our understanding of SM toxicity and accelerate the development of vesicant therapeutics.
We focus on Glutathione S-transferase (GST) and Tissue factor pathway inhibitor-2 (TFPI-2) which closely related with SM, based on the accumulation of our work previously and the research in progress of SM. The interest of this paper is further to discover the role of GST in apoptosis and to acquire TFPI-2 in vitro, and to know about the potent pharmacology of TFPI-2 in SM. The results are as follows:
1. Relation between the variation of GST activity and apoptosis induced by SM.
The variation of GST activity is dose- and time-dependent of SM in mouse ear model. GST activity decreased significantly at 48h post exposure, and also decreased at the dose 160mg/ml SM. It suggested that the skin can’t scavenge free radicals and sulfur mustard at these conditions. The result is also in accordance with the proteomic analysis, confirmed with the decrease of GST after exposure of SM.
We explored the condition of primary fibroblast apoptotic induced by SM.A large number of fibroblast apoptotic were observed in a study of exposure to 100μM SM at 16h, GST activity decreased at the same condition. This result is consistent with our previous findings that the decrease in the expression of GST might relate to epidermal basal cells apoptosis (not published).
2. Effects of increased GST on apoptosis induced by SM.
We obtain GST gene by PCR from a mouse liver, directly insert GST gene into plasmid pCMV, and successfully transfect GST gene into HaCaT by lipofectamine. The cell expressed exogenous GST gene was named as HaCaT/GST.
The percentage of apoptosis cell decreased in HaCaT/GST compared with HaCaT at 16h post exposure to 100μM SM. Such investigation may confirm that GST participates in the apoptosis in SM, and can alleviate the toxicity of SM.
The activity of SOD, the GSH and MDA level were measured at 6h, 12h and 24h after SM administration. The results show that both SOD activity and GSH level were significantly decreased after post-exposure, while the MDA level was enhanced in HaCaT. These results suggested that endogenous antioxidant system was interrupted by the excess free radical production and lipid per-oxidation. So SM induced apoptosis may be associated with the antioxidant defense systems interruption, and the GSH may also have relation with this progress.
3. Molecular cloning, prokaryotic expression and purification of TFPI-2 and the first Kunitz-type domain of TFPI-2.
The TFPI-2, obtained by PCR from a women placenta, was directly inserted into expression vector. And the first Kunitz-type domain of TFPI-2 (TFPI-2/KD1), screened in TFPI-2, was also cloned into expression vector. After cell expression, TFPI-2 expressed as inclusion bodies, while TFPI-2/KD1 exist in supernatant. These results implied that TFPI-2/KD1 in E coli has biological activity. Purification of recombinant TFPI-2/KD1 was performed using His-tag.
4. Biological function of TFPI-2/KD1.
4.1 Biological activity assay of TFPI-2/KD1.
Subsequently TFPI-2/KD1 inhibits the activity of MMPs. Chromogenic and gelatin zymography methods were used to evaluate the inhibiting effects of purified KD1 on trypsin and MMPs individually.
The result suggested that the protein can inhibit MMP-2 and MMP-9 at 20μM of TFPI-2/KD1, and dose-dependently inhibit trypsin. This result implied that TFPI-2/KD1 can inhibit serine protease.
4.2 Effects of TFPI-2/KD1 on inflammatory cytokines release after SM exposure.
The release of inflammatory cytokines including interleukin-8 (IL-8), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) significantly increased at 8h after exposure of 100μM SM in HaCaT. At the dose of 1mg/mL and 2mg/mL TFPI-2/KD1 can significantly suppress SM-increased IL-8 and IL-6 can also be inhibited when administrate 1mg/ml TFPI-2/KD1. But TFPI-2/KD1 has no effect on TNF-α. The fact that TFPI-2/KD1 can inhibit the production of inflammatory cytokines suggested it can at least partly alleviate the toxicity of SM.
4.3 Changes of skin blood perfusion and histopathology induced by SM.
To assessment the lesion depth of different dose SM-exposure (1μl, 2.5μl, 5μl, 10μl); the dermal blood flow was studied by laser Doppler perfusion imaging (LDPI) for three days. And after 72h post exposure, the skins were processed for routine H & E histological evaluation to determine the lesion.
Blood flow of the damage areas became sufficient in a short time. By the time going on the blood flow of damaged skin reduced, and around it circulation increased. This phenomenon was dose- and time- dependent. The result suggested the subcutaneous’vascular were damaged.
The skin biopsies showed that the normal rabbit skin composed with epidermis, dermis, and subcutaneous tissue. A biopsy from 1μl SM exposed skin showed epidermal erosion, ulceration, sub-epidermal bullous (>5mm), hair follicles and sebaceous glands hyperplasia. Along with the increased poison dose, part of the epidermis disappear, necrosis, exudative; vasodilator and congestive in dermis; inflammatory cell infiltration into adnexa, covered the acute inflammatory response. At the dose of 5μl SM, biopsies showed epidermis focal necrosis and obvious dermal hemorrhage.
4.4 Intervention roles of TFPI-2/KD1 on skin inflammation induced by SM. TFPI-2/KD1 has no effect on the blood flow, which can reflect the degree of SM induced inflammatory. H & E histological evaluation suggested that TFPI-2/KD1 can partly alleviate the inflammation response to SM, such as the reduction of neutrophil infiltrating. This indicated that single drug can’t effectively alleviate the SM toxicity on skin.
In summary, GST and TFPI-2/KD1 may play intervention roles in SM skin damage by different pathways. The former is correlated with the apoptosis and the later can partly against with the acute inflammation. Whereas SM has multiple stratification and multiple targets, the mitigation of single drug is limited, we must seek for the key mediator and take the multiple drug/agent medical countermeasures.
自芥子气出现于第一次世界大战以来,针对芥子气防护以及损伤治疗的相关研究一直持续,并向深入发展。但是,迄今为止,芥子气毒理学机制仍未完全阐明,针对芥子气中毒的特效抗毒药物仍然属于空白,临床上仅能采取对症治疗措施,究其原因,可能与芥子气作为双功能烃化剂,其毒理学作用具有多水平、多靶点的特点有关。因此,芥子气皮肤损伤毒理学机制以及基于相关研究基础之上的特效抗毒药物研究策略的探索和有效实施,仍是国内外芥子气研究领域关注的工作重点之一。
基于课题组前期工作积累,并结合国外研究进展,本课题选取了与芥子气皮肤损伤具有密切关系的谷胱甘肽硫转移酶(glutathione-S-transferase, GST),以及组织因子途径抑制剂-2 (tissue factor pathway inhibitor-2, TFPI-2)作为研究重点,旨在进一步了解GST在芥子气诱导细胞凋亡中的作用,探索其能否成为缓解芥子气毒性的新途径,同时,利用生物工程技术体外制备TFPI-2,探讨其作为一种丝氨酸蛋白酶抑制剂,在芥子气损伤预防和治疗研究中的价值,并试图通过这种关注和积累芥子气皮肤损伤不同环节作用资料的研究,以逐步对特效抗毒药物研究和开发策略的制定提供有意义的帮助。
1.芥子气诱导GST活性变化规律与细胞凋亡情况的关系研究。
在芥子气皮肤损伤小鼠耳廓模型上,GST活性变化具有剂量依赖性和时间依赖性。随着芥子气染毒剂量加大和染毒时间延长,GST活性下降,至染毒剂量为160mg/ml时所有时间点,GST活性显著下降;所有剂量组染毒后72h,GST活性显著下降,与课题组前期蛋白质组学实验结果取得了较好的一致性,明显提示,芥子气染毒后,皮肤组织依赖于GST的对自由基及芥子气的清除能力有所减弱。
将芥子气作用于原代培养的乳鼠皮肤成纤维细胞,观察由其诱导细胞发生凋亡的情况,结果发现,在芥子气浓度为100μM染毒后16h,细胞凋亡明显,此时细胞内GST活性显著下降,至芥子气染毒后24h,死亡细胞显著增多。结果提示芥子气诱导的细胞凋亡与GST活性降低在某种程度上可能具有关联性。
2.增加细胞内GST对芥子气诱导细胞凋亡效应的影响
从GST含量丰富的小鼠肝脏提取总RNA,经过PCR扩增反应,测序并与NCBI序列库比对,证实取得正确的GST基因,将该基因与pCMV真核表达载体进行载体构建,为后续细胞转染奠定基础。
通过脂质体转染技术,将携带有克隆基因片段GST的真核表达载体转染至HaCaT细胞系,并利用免疫印迹对其表达进行验证,将该细胞称为HaCaT/GST。芥子气染毒两种细胞(染毒浓度为100μM,检测时间为16h),HaCaT/GST与HaCaT细胞相比,细胞凋亡百分比明显下降,证实了单独转染GST对于芥子气诱导的细胞凋亡有防护和对抗作用。
在上述染毒条件下,分别于染毒后6h、12h、24h,检测HaCaT细胞内GSH、SOD和MDA变化情况。结果表明,随着芥子气染毒时间延长,GSH含量降低,脂质过氧化反应产物MDA含量逐渐增加,SOD被芥子气刺激产生的自由基所耗竭,含量逐渐下降,细胞内氧化还原平衡被破坏,最终引起细胞损伤。提示芥子气诱导的细胞凋亡与氧化还原系统失衡有关,并且GSH耗竭在该过程中可能起一定作用。
3.TFPI-2分子克隆及其第一结构域的基因克隆、蛋白表达与纯化为了探讨TFPI-2能否缓解芥子气毒性,从TFPI-2含量较高的人胎盘调取该基因,并从其全长基因中将含有Kunitz结构域1(first Kunitz-type domain, KD1),即TFPI-2/KD1基因片段进行扩增。分别将TFPI-2和TFPI-2/KD1构建至原核表达载体,利用原核表达的方法将两种蛋白进行体外表达,结果表明TFPI-2以包涵体形式表达,而TFPI-2/KD1以可溶的形式表达于细胞内上清液,提示TFPI-2/KD1可能具有生物活性,不需经过复性过程。因此,在后续的实验研究中,我们对TFPI-2/KD1进行了纯化,进一步探讨其活性和功能。
4.TFPI-2/KD1的功能研究
4.1 TFPI-2/KD1的活性验证
采用明胶酶谱法检测TFPI-2/KD1能否抑制H1299癌细胞株分泌的基质金属蛋白酶;采用发色底物法检测TFPI-2/KD1能否抑制胰蛋白酶。结果表明,TFPI-2 /KD1浓度为20μM时能够显著的抑制MMP-2、MMP-9;在一定浓度范围内,随TFPI-2/KD1浓度的升高,对胰蛋白酶的抑制作用呈现增强的趋势。提示TFPI-2 /KD1具有抑制丝氨酸蛋白酶的活性。
4.2 TFPI-2/KD1对于芥子气诱导炎症因子分泌的影响
采用放射免疫分析法检测炎症因子IL-6、IL-8和TNF-α。与正常细胞相比较,100μM芥子气染毒HaCaT细胞后8h,上清液中IL-6、IL-8和TNF-α均显著分泌。TFPI-2/KD1蛋白为1mg/ml以及2mg/ml时,能够显著性抑制芥子气诱导IL-8分泌;TFPI-2/KD1蛋白浓度为1mg/ml时,能够显著性抑制芥子气诱导IL-6分泌;而各浓度组TFPI-2/KD1(0.5mg/ml、1mg/ml以及2mg/ml)对于芥子气诱导的TNF-α分泌升高却没有抑制作用。基于以上结果,我们推测TFPI-2/KD1通过影响细胞因子分泌,可能具有对抗芥子气诱导炎症反应的作用。
4.3芥子气染毒家兔皮肤血流变化与病理损伤研究
利用激光多普勒仪检测不同剂量芥子气(1μl、2.5μl、5μl、10μl)染毒家兔背部皮肤血流变化情况,连续观察至染毒后第三天,将动物处死,做石蜡切片检测皮肤的病理损伤程度。
结果显示,芥子气染毒后,染毒皮肤短时间内即出现血流加快,提示染毒部位可能出现急性炎症反应,血管扩张;随着时间的延长,中心区域供血减弱,出现环形供血区域,提示染毒部位的血管可能产生损伤,周围组织代偿性供血。随着染毒时间延长和剂量增加,环形供血区域更加明显。
病理切片结果显示,家兔正常背部皮肤可见表皮、真皮、附属器及肌肉组织;1μl芥子气染毒后表皮出现糜烂、溃疡、表皮下大疱(>5mm)、毛囊和皮脂腺有瘤样增生;随着染毒剂量加大,部分表皮消失,表皮可见坏死及渗出物;真皮层血管扩张、充血、水肿,有大量的炎细胞浸润并深达附属器;急性炎症反应明显(中性粒细胞为主);染毒剂量为10μl时,表皮出现灶性坏死,出血明显。
4.4 TFPI-2/KD1干预芥子气引起皮肤炎症的初步探讨
TFPI-2/KD1(给药剂量为1mg/ml,4次/日)对伴随芥子气急性损伤时所观察到的,体现急性炎症性反应特点的血液流变规律没有明显影响;病理结果显示,与对照组比较,TFPI-2/KD1给药后染毒皮肤炎症反应有所减轻,表现为中性粒细胞浸润数量减少,表皮灶性坏死程度减轻。但总体上没能非常有效地改善芥子气染毒所致皮肤病理损伤,芥子气染毒皮肤表面出血、灶性的炎症反应等病理变化仍十分常见。提示TFPI-2/KD1对芥子气所致皮肤损伤治疗效果有限,而这一现象在芥子气的治疗中具有普遍提示性:单一药物干预效果不理想。
简而言之,在芥子气皮肤损伤过程中GST以及TFPI-2通过不同作用环节起到相应的作用,前者主要与芥子气诱导的细胞凋亡相关,而后者可能通过对丝氨酸蛋白酶的抑制效应,部分地对抗了芥子气诱导的急性炎症反应。但是,单一途径的干预效果仍然十分有限,体现了芥子气损伤的多部位、多靶点特征,提示针对芥子气损伤特效抗毒药物研究和开发策略不应仅局限于单一环节,而应该努力寻找最为关键的环节和靶点,并尽量兼顾其多部位、多靶点特征。
Sulfur mustard (SM),“the king of the chemical warfare agents,”is a typical vesicant agent. Skin is the major target for SM., the most prominent toxic effects of which are extremely slow healing lesions and blisters resulting in ulcer and vesicle of the skin, and promoting secondary infections.
Although the toxicity of SM has been investigated since it was used in the World War I. Presently, the mechanisms underlying sulfur mustard-induced skin damage are not fully elucidated and no antidote exists, apart from its characteristics of multiple stratification and multiple targets. Current treatment strategy is designed to relieve symptoms. Critical questions are proposed to increase our understanding of SM toxicity and accelerate the development of vesicant therapeutics.
We focus on Glutathione S-transferase (GST) and Tissue factor pathway inhibitor-2 (TFPI-2) which closely related with SM, based on the accumulation of our work previously and the research in progress of SM. The interest of this paper is further to discover the role of GST in apoptosis and to acquire TFPI-2 in vitro, and to know about the potent pharmacology of TFPI-2 in SM. The results are as follows:
1. Relation between the variation of GST activity and apoptosis induced by SM.
The variation of GST activity is dose- and time-dependent of SM in mouse ear model. GST activity decreased significantly at 48h post exposure, and also decreased at the dose 160mg/ml SM. It suggested that the skin can’t scavenge free radicals and sulfur mustard at these conditions. The result is also in accordance with the proteomic analysis, confirmed with the decrease of GST after exposure of SM.
We explored the condition of primary fibroblast apoptotic induced by SM.A large number of fibroblast apoptotic were observed in a study of exposure to 100μM SM at 16h, GST activity decreased at the same condition. This result is consistent with our previous findings that the decrease in the expression of GST might relate to epidermal basal cells apoptosis (not published).
2. Effects of increased GST on apoptosis induced by SM.
We obtain GST gene by PCR from a mouse liver, directly insert GST gene into plasmid pCMV, and successfully transfect GST gene into HaCaT by lipofectamine. The cell expressed exogenous GST gene was named as HaCaT/GST.
The percentage of apoptosis cell decreased in HaCaT/GST compared with HaCaT at 16h post exposure to 100μM SM. Such investigation may confirm that GST participates in the apoptosis in SM, and can alleviate the toxicity of SM.
The activity of SOD, the GSH and MDA level were measured at 6h, 12h and 24h after SM administration. The results show that both SOD activity and GSH level were significantly decreased after post-exposure, while the MDA level was enhanced in HaCaT. These results suggested that endogenous antioxidant system was interrupted by the excess free radical production and lipid per-oxidation. So SM induced apoptosis may be associated with the antioxidant defense systems interruption, and the GSH may also have relation with this progress.
3. Molecular cloning, prokaryotic expression and purification of TFPI-2 and the first Kunitz-type domain of TFPI-2.
The TFPI-2, obtained by PCR from a women placenta, was directly inserted into expression vector. And the first Kunitz-type domain of TFPI-2 (TFPI-2/KD1), screened in TFPI-2, was also cloned into expression vector. After cell expression, TFPI-2 expressed as inclusion bodies, while TFPI-2/KD1 exist in supernatant. These results implied that TFPI-2/KD1 in E coli has biological activity. Purification of recombinant TFPI-2/KD1 was performed using His-tag.
4. Biological function of TFPI-2/KD1.
4.1 Biological activity assay of TFPI-2/KD1.
Subsequently TFPI-2/KD1 inhibits the activity of MMPs. Chromogenic and gelatin zymography methods were used to evaluate the inhibiting effects of purified KD1 on trypsin and MMPs individually.
The result suggested that the protein can inhibit MMP-2 and MMP-9 at 20μM of TFPI-2/KD1, and dose-dependently inhibit trypsin. This result implied that TFPI-2/KD1 can inhibit serine protease.
4.2 Effects of TFPI-2/KD1 on inflammatory cytokines release after SM exposure.
The release of inflammatory cytokines including interleukin-8 (IL-8), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) significantly increased at 8h after exposure of 100μM SM in HaCaT. At the dose of 1mg/mL and 2mg/mL TFPI-2/KD1 can significantly suppress SM-increased IL-8 and IL-6 can also be inhibited when administrate 1mg/ml TFPI-2/KD1. But TFPI-2/KD1 has no effect on TNF-α. The fact that TFPI-2/KD1 can inhibit the production of inflammatory cytokines suggested it can at least partly alleviate the toxicity of SM.
4.3 Changes of skin blood perfusion and histopathology induced by SM.
To assessment the lesion depth of different dose SM-exposure (1μl, 2.5μl, 5μl, 10μl); the dermal blood flow was studied by laser Doppler perfusion imaging (LDPI) for three days. And after 72h post exposure, the skins were processed for routine H & E histological evaluation to determine the lesion.
Blood flow of the damage areas became sufficient in a short time. By the time going on the blood flow of damaged skin reduced, and around it circulation increased. This phenomenon was dose- and time- dependent. The result suggested the subcutaneous’vascular were damaged.
The skin biopsies showed that the normal rabbit skin composed with epidermis, dermis, and subcutaneous tissue. A biopsy from 1μl SM exposed skin showed epidermal erosion, ulceration, sub-epidermal bullous (>5mm), hair follicles and sebaceous glands hyperplasia. Along with the increased poison dose, part of the epidermis disappear, necrosis, exudative; vasodilator and congestive in dermis; inflammatory cell infiltration into adnexa, covered the acute inflammatory response. At the dose of 5μl SM, biopsies showed epidermis focal necrosis and obvious dermal hemorrhage.
4.4 Intervention roles of TFPI-2/KD1 on skin inflammation induced by SM. TFPI-2/KD1 has no effect on the blood flow, which can reflect the degree of SM induced inflammatory. H & E histological evaluation suggested that TFPI-2/KD1 can partly alleviate the inflammation response to SM, such as the reduction of neutrophil infiltrating. This indicated that single drug can’t effectively alleviate the SM toxicity on skin.
In summary, GST and TFPI-2/KD1 may play intervention roles in SM skin damage by different pathways. The former is correlated with the apoptosis and the later can partly against with the acute inflammation. Whereas SM has multiple stratification and multiple targets, the mitigation of single drug is limited, we must seek for the key mediator and take the multiple drug/agent medical countermeasures.
引文
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