益母草抗皮肤光老化的实验研究
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摘要
目的：
本项研究依据中医药基础理论,运用生化分析及免疫组化等方法,观察紫外线照射引起的小鼠皮肤光老化模型血清及皮肤组织中SOD、GSH-Px和CAT的活性；H2O2、MDA以及HYP含量的变化；Bax、Bcl-2、TGF-β1蛋白表达水平的变化；皮肤组织结构的变化,以及中药益母草的干预作用及其可能的作用机制。
材料与方法：
165只清洁级昆明种45日龄小鼠,体重18±2.2g,均为雌性,随机分成11组：正常对照组(A)、模型对照组(B)、模型对照组(C)、‘阳性药物预防组(D)、外用益母草低剂量预防组(E)、外用益母草中剂量预防组(F)、外用益母草高剂量预防组(G)、灌胃益母草低剂量预防组(H)、灌胃益母草中剂量预防组(I)、灌胃益母草高剂量预防组(J)、治疗组(K)。A组小鼠正常饲养,不做任何处理。从实验开始至实验结束(第1周至第13周末),B组小鼠每日背部涂抹生理盐水0.4ml；C组小鼠每日经口灌胃0.4ml生理盐水；D组小鼠每日背部涂抹VitE一粒；E、F、G组为外用药预防组,给予益母草赋形剂进行背部皮肤外涂预防(浓度分别为4%、8%、16%)；H、I、J组为经口灌胃预防组,每日经口灌胃0.4ml相应浓度的益母草进行预防(用药量分别为0.78、1.56、3.12g生药/kg,相当于益母草煎液浓度0.039、0.078、0.156g/ml),以上各组于第3周起,在给药30min之后,进行紫外线照射；K组小鼠实验前2周不作任何处理,第3周起先给予紫外线照射,照射结束30min之后,外涂中剂量益母草进行治疗。第14周起终止紫外线照射,继续进行益母草外涂治疗2周。HE染色,光镜下观察皮肤组织结构的改变,并用生化分析方法测定SOD、CAT和GSH-Px的活性,MDA、H2O以及HYP的含量,免疫组化法检测Bax、Bcl-2、TGF-β1蛋白表达水平的变化情况。
结果：
1.动物造模实验中,光老化小鼠皮肤组织中SOD、CAT和GSH-Px活性明显降低(P<0.01),MDA、H2O含量显著增加(P<0.01)；HYP含量明显减少(P<0.01)；皮肤组织病理切片呈现光老化状态。
2.外用益母草各剂量预防组与模型对照组B比较,灌胃益母草各剂量预防组与模型对照组C比较,外用及灌胃各剂量组小鼠皮肤组织中HYP含量均有明显升高,差异有统计学意义(P<0.05,P<0.01)；组间比较显示：外用低剂量组与灌胃低剂量组HYP含量比较,差异有统计学意义(P<0.05)；外用中剂量组与灌胃中剂量组HYP含量比较,无统计学意义(P>0.05)；外用高剂量组与灌胃高剂量组HYP含量比较,差异有统计学意义(P<0.05)。
3.外用益母草各剂量预防组与模型对照组B比较,灌胃益母草各剂量预防组与模型对照组C比较,外用及灌胃各剂量组小鼠皮肤组织中MDA含量下降,SOD活力增高,具有显著性差异(P<0.01)。组间比较显示：外用低剂量组与灌胃低剂量组MDA含量比较,差异有统计学意义(P<0.01)；外用中剂量组与灌胃中剂量组MDA含量比较,无统计学意义(P>0.05)：外用高剂量组与灌胃高剂量组MDA含量比较,差异有统计学意义(P<0.05)。外用低、中、高剂量组分别与灌胃低、中、高剂量组SOD活力比较,无统计学意义(P>0.05)。
4.外用益母草各剂量预防组与模型对照组B比较,灌胃益母草各剂量预防组与模型对照组C比较,外用及灌胃益母草各剂量组小鼠皮肤组织中CAT活力提高、GSH-Px活力提高、H2O2含量降低,差异有统计学意义(P<0.05,P<0.01)。组间比较显示：外用低、中、高剂量组分别与灌胃低、中、高剂量组CAT活力比较,无统计学意义(P>0.05)。外用中、高剂量组与灌胃中、高剂量组GSH-Px活力比较,差异有统计学意义(P<0.05)。外用中、高剂量组与灌胃中、高剂量组H2O2含量比较,无统计学意义(P>0.05)。
5.外用益母草各剂量预防组与模型对照组B比较,灌胃益母草各剂量预防组与模型对照组C比较,外用及灌胃益母草各剂量组小鼠皮肤组织中Bax蛋白表达下调、Bcl-2蛋白表达上调、TGF-β1蛋白表达上调,差异有统计学意义(P<0.01)。组间比较显示：外用低、中、高剂量组分别与灌胃低、中、高剂量组Bax比较,无统计学意义(P>0.05)。外用高剂量组与灌胃高剂量组Bcl-2蛋白表达水平比较,差异有统计学意义(P<0.01)。外用高剂量组与灌胃高剂量组TGF-β1蛋白表达水平比较,差异有统计学意义(P<0.01)。
6.与模型对照组B比较,治疗组(K组)小鼠皮肤组织中SOD、CAT、GSH-Px活性增强,差异有统计学意义(P<0.01)；H2O2、MDA含量降低,差异有统计学意义(P<0.01)；HYP含量提高,差异有统计学意义(P<0.01)；Bcl-2蛋白表达上调,Bax蛋白表达下调,TGF-β1蛋白表达上调,差异有统计学意义(P<0.01)。
结论：
1.利用紫外线的长期照射可成功复制皮肤光老化动物模型。紫外线照射所致小鼠皮肤光老化,其皮肤组织有明显的氧化损伤,皮肤组织中细胞凋亡加速。
2.外用益母草低中高剂量预防组疗效有差异；灌胃益母草低中高剂量预防组疗效无差异。益母草外用疗效优于灌胃。
3.益母草可通过清除自由基；提高机体抗氧化能力、提高酶活力；减轻小鼠皮肤组织的氧化损伤,促进胶原蛋白合成,减缓细胞凋亡,从而预防皮肤光老化。益母草对皮肤光老化的修复作用可能与促使TGF-β1上调,从而促进胶原蛋白合成有关。
4.益母草亦能够修复小鼠光老化皮肤的组织损伤。
Objective:The experiment is according as the basic theory of TCM, to investigate LSH's influence on the activity of SOD, GSH-Px and CAT in the skin of photoaging mice, wich result from ultraviolet irradiation, as well as the content of HA, MDA and HYP in their skin, the expression levels of Bcl-2protein、Bax and TGF-β1; and to investigate LSH's effect on the structure of the photoaging mice. To establish the animal model of skin photoaging as well as to study the protective effects of LSH.
Methods:Healthy KM mice were divided into11groups randomly and respective-ly,45days age, weight18+2.2g, female. Falls into negative control group (A), external-model group(B), oral-model group(C), positive druggery defending group(D), external low-LHS-dosage preventing group(E), external medium-LHS-dosage preventing group(F), external high-LHS-dosage preventing group(G), oral low-LHS-dosage preventing group(H), oral medium-LHS-dosage preventing group (I), oral high-LHS-dosage preventing group (J), external medium-LHS-dosage preventing group(afer UV irradiation)(K). The mice in all groups but group A were irradiated to simulate skin photoaging by long-term ultraviolet irradiation.30minutes before UV irradiation, external0.9%physilological saline,0.4ml to B Group;Oral0.9%physiological saline,20ml/kg to C group;external VitE to D Group;At the same time, the mice those in E, F, and G groups were given LHS externally and which in H, I, J groups through mouth. Staining skin with HE, under40times light microscope, the structure of dermal was observed. While the changes of the content of H2O2, MDA, HYP, as well as the activity of SOD, GSH-Px and CAT, the changes of the expression levels of Bax, Bcl-2and TGF-β1in the skin were measured by biochemical test method in each group of the experiment.
Results:
1. In model group, the activity of SOD, CAT and GSH-Px in dermal were decreased(p <0.01), while the contents of the MDA, H2O2were increased seriously (P<0.01). on the contrary, the content of HYP was reduced (P<0.01). The skin biopsies appear photoaging look.
2. Compared with the model group, the contents of HYP in external and oral groups were made a great progress (P<0.01, P<0.05); It can make differences with the contents of HYP in low-LHS-dosage external and oral groups (P<0.05);midium-LHS-dosage external and oral groups(P<0.05). It can make any differences with the contents of HYP in low-LHS-dosage external and high-LHS-dosage oral groups (P>0.05).
3. Compared with the model group, in external and oral groups, the contents of MDA were decreased, the SOD's energy were elevation (P<0.01). The contents of MDA in external and oral groups were made a great progress (P<0.01);It can make any differences with the contents of MDA in medium-LHS-dosage external and oral groups (P>0.05). It can make differences with the contents of MDA in high-LHS-dosage external and oral groups (P<0.05);The SOD's energy were make any sense both in the external and oral three groups(P>0.05).
4.Compared with the model group, the activity of CAT's, GSH-Px's energy were elevation, the contents of HA were decreased (P<0.05, P<0.01). The CAT's energy were make any sense both in the external and oral three groups(P>0.05). The contents of HA were make any sense both in the medium, high-external and oral groups (P>0.05).
5. Compared with the model group, the expression of Bax was down-regulation, Bcl-2was up-regulation, TGF-β1was up-regulation(P<0.01). The expression of Bax was make any sense both in the external and oral three groups (P>0.05). The expression of Bcl-2was make sense between the high-dosage external and oral groups(P<0.01). The expression of TGF-β1was make sense between the high-dosage external and oral groups (P<0.01).
6. Compared with the model group (B), the energy of SOD, CAT, GSH-Px were increased (P <0.01);The contents of HA, MDA were decreased (P<0.01);the contents of HYP was increased(P<0.01);The expression of Bcl-2was up-regulation, Bax was down-regulation, TGF-β1was up-regulation (P<0.01).
Conclusions:
1. Long-term ultraviolet irradiation can make skin of mice become photoaging. UV-irradiation induced photoaging of mice, the derma tissue of mice have oxidative damage obviously, the cells apoptosis were accelerate.
2. LHS can improve the pathological film of mice who have photoaging damage. It has discrepancy in three external groups, It has no-discrepancy in three oral groups (P>0.05). The external groups have therapy effect than oral-groups.
3. LHS could eliminate free radicals itself, and by increasing the ability of inhibiting the oxidative damages of the organism,, remit the trend of cell apoptosis to prevent photoaging damage.
4. LHS can also repair the photoaging damage of the mice derma tissue.
本项研究依据中医药基础理论,运用生化分析及免疫组化等方法,观察紫外线照射引起的小鼠皮肤光老化模型血清及皮肤组织中SOD、GSH-Px和CAT的活性；H2O2、MDA以及HYP含量的变化；Bax、Bcl-2、TGF-β1蛋白表达水平的变化；皮肤组织结构的变化,以及中药益母草的干预作用及其可能的作用机制。
材料与方法：
165只清洁级昆明种45日龄小鼠,体重18±2.2g,均为雌性,随机分成11组：正常对照组(A)、模型对照组(B)、模型对照组(C)、‘阳性药物预防组(D)、外用益母草低剂量预防组(E)、外用益母草中剂量预防组(F)、外用益母草高剂量预防组(G)、灌胃益母草低剂量预防组(H)、灌胃益母草中剂量预防组(I)、灌胃益母草高剂量预防组(J)、治疗组(K)。A组小鼠正常饲养,不做任何处理。从实验开始至实验结束(第1周至第13周末),B组小鼠每日背部涂抹生理盐水0.4ml；C组小鼠每日经口灌胃0.4ml生理盐水；D组小鼠每日背部涂抹VitE一粒；E、F、G组为外用药预防组,给予益母草赋形剂进行背部皮肤外涂预防(浓度分别为4%、8%、16%)；H、I、J组为经口灌胃预防组,每日经口灌胃0.4ml相应浓度的益母草进行预防(用药量分别为0.78、1.56、3.12g生药/kg,相当于益母草煎液浓度0.039、0.078、0.156g/ml),以上各组于第3周起,在给药30min之后,进行紫外线照射；K组小鼠实验前2周不作任何处理,第3周起先给予紫外线照射,照射结束30min之后,外涂中剂量益母草进行治疗。第14周起终止紫外线照射,继续进行益母草外涂治疗2周。HE染色,光镜下观察皮肤组织结构的改变,并用生化分析方法测定SOD、CAT和GSH-Px的活性,MDA、H2O以及HYP的含量,免疫组化法检测Bax、Bcl-2、TGF-β1蛋白表达水平的变化情况。
结果：
1.动物造模实验中,光老化小鼠皮肤组织中SOD、CAT和GSH-Px活性明显降低(P<0.01),MDA、H2O含量显著增加(P<0.01)；HYP含量明显减少(P<0.01)；皮肤组织病理切片呈现光老化状态。
2.外用益母草各剂量预防组与模型对照组B比较,灌胃益母草各剂量预防组与模型对照组C比较,外用及灌胃各剂量组小鼠皮肤组织中HYP含量均有明显升高,差异有统计学意义(P<0.05,P<0.01)；组间比较显示：外用低剂量组与灌胃低剂量组HYP含量比较,差异有统计学意义(P<0.05)；外用中剂量组与灌胃中剂量组HYP含量比较,无统计学意义(P>0.05)；外用高剂量组与灌胃高剂量组HYP含量比较,差异有统计学意义(P<0.05)。
3.外用益母草各剂量预防组与模型对照组B比较,灌胃益母草各剂量预防组与模型对照组C比较,外用及灌胃各剂量组小鼠皮肤组织中MDA含量下降,SOD活力增高,具有显著性差异(P<0.01)。组间比较显示：外用低剂量组与灌胃低剂量组MDA含量比较,差异有统计学意义(P<0.01)；外用中剂量组与灌胃中剂量组MDA含量比较,无统计学意义(P>0.05)：外用高剂量组与灌胃高剂量组MDA含量比较,差异有统计学意义(P<0.05)。外用低、中、高剂量组分别与灌胃低、中、高剂量组SOD活力比较,无统计学意义(P>0.05)。
4.外用益母草各剂量预防组与模型对照组B比较,灌胃益母草各剂量预防组与模型对照组C比较,外用及灌胃益母草各剂量组小鼠皮肤组织中CAT活力提高、GSH-Px活力提高、H2O2含量降低,差异有统计学意义(P<0.05,P<0.01)。组间比较显示：外用低、中、高剂量组分别与灌胃低、中、高剂量组CAT活力比较,无统计学意义(P>0.05)。外用中、高剂量组与灌胃中、高剂量组GSH-Px活力比较,差异有统计学意义(P<0.05)。外用中、高剂量组与灌胃中、高剂量组H2O2含量比较,无统计学意义(P>0.05)。
5.外用益母草各剂量预防组与模型对照组B比较,灌胃益母草各剂量预防组与模型对照组C比较,外用及灌胃益母草各剂量组小鼠皮肤组织中Bax蛋白表达下调、Bcl-2蛋白表达上调、TGF-β1蛋白表达上调,差异有统计学意义(P<0.01)。组间比较显示：外用低、中、高剂量组分别与灌胃低、中、高剂量组Bax比较,无统计学意义(P>0.05)。外用高剂量组与灌胃高剂量组Bcl-2蛋白表达水平比较,差异有统计学意义(P<0.01)。外用高剂量组与灌胃高剂量组TGF-β1蛋白表达水平比较,差异有统计学意义(P<0.01)。
6.与模型对照组B比较,治疗组(K组)小鼠皮肤组织中SOD、CAT、GSH-Px活性增强,差异有统计学意义(P<0.01)；H2O2、MDA含量降低,差异有统计学意义(P<0.01)；HYP含量提高,差异有统计学意义(P<0.01)；Bcl-2蛋白表达上调,Bax蛋白表达下调,TGF-β1蛋白表达上调,差异有统计学意义(P<0.01)。
结论：
1.利用紫外线的长期照射可成功复制皮肤光老化动物模型。紫外线照射所致小鼠皮肤光老化,其皮肤组织有明显的氧化损伤,皮肤组织中细胞凋亡加速。
2.外用益母草低中高剂量预防组疗效有差异；灌胃益母草低中高剂量预防组疗效无差异。益母草外用疗效优于灌胃。
3.益母草可通过清除自由基；提高机体抗氧化能力、提高酶活力；减轻小鼠皮肤组织的氧化损伤,促进胶原蛋白合成,减缓细胞凋亡,从而预防皮肤光老化。益母草对皮肤光老化的修复作用可能与促使TGF-β1上调,从而促进胶原蛋白合成有关。
4.益母草亦能够修复小鼠光老化皮肤的组织损伤。
Objective:The experiment is according as the basic theory of TCM, to investigate LSH's influence on the activity of SOD, GSH-Px and CAT in the skin of photoaging mice, wich result from ultraviolet irradiation, as well as the content of HA, MDA and HYP in their skin, the expression levels of Bcl-2protein、Bax and TGF-β1; and to investigate LSH's effect on the structure of the photoaging mice. To establish the animal model of skin photoaging as well as to study the protective effects of LSH.
Methods:Healthy KM mice were divided into11groups randomly and respective-ly,45days age, weight18+2.2g, female. Falls into negative control group (A), external-model group(B), oral-model group(C), positive druggery defending group(D), external low-LHS-dosage preventing group(E), external medium-LHS-dosage preventing group(F), external high-LHS-dosage preventing group(G), oral low-LHS-dosage preventing group(H), oral medium-LHS-dosage preventing group (I), oral high-LHS-dosage preventing group (J), external medium-LHS-dosage preventing group(afer UV irradiation)(K). The mice in all groups but group A were irradiated to simulate skin photoaging by long-term ultraviolet irradiation.30minutes before UV irradiation, external0.9%physilological saline,0.4ml to B Group;Oral0.9%physiological saline,20ml/kg to C group;external VitE to D Group;At the same time, the mice those in E, F, and G groups were given LHS externally and which in H, I, J groups through mouth. Staining skin with HE, under40times light microscope, the structure of dermal was observed. While the changes of the content of H2O2, MDA, HYP, as well as the activity of SOD, GSH-Px and CAT, the changes of the expression levels of Bax, Bcl-2and TGF-β1in the skin were measured by biochemical test method in each group of the experiment.
Results:
1. In model group, the activity of SOD, CAT and GSH-Px in dermal were decreased(p <0.01), while the contents of the MDA, H2O2were increased seriously (P<0.01). on the contrary, the content of HYP was reduced (P<0.01). The skin biopsies appear photoaging look.
2. Compared with the model group, the contents of HYP in external and oral groups were made a great progress (P<0.01, P<0.05); It can make differences with the contents of HYP in low-LHS-dosage external and oral groups (P<0.05);midium-LHS-dosage external and oral groups(P<0.05). It can make any differences with the contents of HYP in low-LHS-dosage external and high-LHS-dosage oral groups (P>0.05).
3. Compared with the model group, in external and oral groups, the contents of MDA were decreased, the SOD's energy were elevation (P<0.01). The contents of MDA in external and oral groups were made a great progress (P<0.01);It can make any differences with the contents of MDA in medium-LHS-dosage external and oral groups (P>0.05). It can make differences with the contents of MDA in high-LHS-dosage external and oral groups (P<0.05);The SOD's energy were make any sense both in the external and oral three groups(P>0.05).
4.Compared with the model group, the activity of CAT's, GSH-Px's energy were elevation, the contents of HA were decreased (P<0.05, P<0.01). The CAT's energy were make any sense both in the external and oral three groups(P>0.05). The contents of HA were make any sense both in the medium, high-external and oral groups (P>0.05).
5. Compared with the model group, the expression of Bax was down-regulation, Bcl-2was up-regulation, TGF-β1was up-regulation(P<0.01). The expression of Bax was make any sense both in the external and oral three groups (P>0.05). The expression of Bcl-2was make sense between the high-dosage external and oral groups(P<0.01). The expression of TGF-β1was make sense between the high-dosage external and oral groups (P<0.01).
6. Compared with the model group (B), the energy of SOD, CAT, GSH-Px were increased (P <0.01);The contents of HA, MDA were decreased (P<0.01);the contents of HYP was increased(P<0.01);The expression of Bcl-2was up-regulation, Bax was down-regulation, TGF-β1was up-regulation (P<0.01).
Conclusions:
1. Long-term ultraviolet irradiation can make skin of mice become photoaging. UV-irradiation induced photoaging of mice, the derma tissue of mice have oxidative damage obviously, the cells apoptosis were accelerate.
2. LHS can improve the pathological film of mice who have photoaging damage. It has discrepancy in three external groups, It has no-discrepancy in three oral groups (P>0.05). The external groups have therapy effect than oral-groups.
3. LHS could eliminate free radicals itself, and by increasing the ability of inhibiting the oxidative damages of the organism,, remit the trend of cell apoptosis to prevent photoaging damage.
4. LHS can also repair the photoaging damage of the mice derma tissue.
引文
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