| [Objective]1.The mice homozygous and cre positive for Flox of spred-1 gene and homozygous and cre negative for Flox of spred-1 gene were constructed.2.C57BL/6J mouse psoriasis model induced by imiquimod modified modeling method was prepared and evaluated to obtain a psoriasis like mouse model with more lasting time and more typical pathological characteristics.3.Through the study of conditional knockdown of spred-1 gene in psoriasis like mice,to find the exact evidence that spred-1 protein is involved in the occurrence and development of psoriasis.4.Through the intervention effect of qingxuedu granule mixture on spred-1 gene knockdown psoriasis like mouse model,to explore whether spred-1/ERK pathway is the possible mechanism of its treatment of psoriasis.[Method]1.The spred-1 gene was modified by Flox by homologous recombination of fertilized eggs;Krt14-（Cre ERT2）hybrid mice were constructed by typing Cre ERT2-wpre-p A expression frame at the start codon of krt14 gene.Mice were bred by the following two methods:one method was to mate the obtained double positive hybrid mice of Flox and cre of spred-1 gene with Flox hybrid mice;Another method is to mate the obtained spred-1 gene Flox and cre double positive hybrid mice with spred-1 gene Flox homozygous mice.2.C57BL/6J mice were randomly divided into normal group（smeared with Vaseline）and modified group[smeared with imiquimod cream+subcutaneous injection of recombinant mouse interleukin-12 and lipopolysaccharide（once a week）]for 14 days,with 8 mice in each group.3.Thirty two mice were randomly divided into 4 groups:Non inducible spred-1 conditional knockdown mice[Spred-1（+）Cre（-）,Spred-1+/-]weredivided into corn oil group（control group 1）and tamoxifen group（control group 2）,with 8 mice in each group;Conditional knockdown of inducible spred-1 gene in mice[Spred-1（+）Cre（+）,Spred-1+/+]were divided into corn oil group（control group 3）and tamoxifen group（experimental group 4）,with 8 mice in each group.At the beginning of the experiment,spred-1+/-corn oil group（control group 1）and the Spred-1+/+corn oil group（control group 3）were intraperitoneally injected with corn oil（dose120mg/kg/mouse）,the Spred-1+/-tamoxifen group（control group 2）andSpred-1+/+tamoxifen group（experimental group 4）were intraperitoneally injected with tamoxifen（dose 120mg/kg/mouse）,once every other day,5times in total.After gene induced knockdown injection,the back of mice was depilated and the model was established.The mice in the four groups were regularly applied with imiquimod cream and subcutaneously injected with recombinant mouse interleukin-12（rm IL-12）and lipopolysaccharide（once a week）]and killed for 14 days.The changes of PASI score and histopathology of skin lesions were observed;At the same time,Western blot and RT-PCR experiments were carried out to observe the expression of spred-1 protein in non/near lesions,lesions and RAS/ERK signal pathway related protein and m RNA in lesions.4.Thirty two mice were randomly divided into four groups:Spred-1+/-qingxuedu granule group（control group 5）:8 mice;Spred-1+/+qingxuedu granule group（experimental group 6）,Spred-1+/+compound Qingdai capsule group（control group 7）and Spred-1+/+model group（model group 8）,with8 mice in each group.The tail was cut on day 0,day 10 and day 24 to detect the expression of spred-1 gene.On the first day of the experiment,the Spred-1+/-qingxuedu granule group（control group 5）,Spred-1+/+qingxuedu granule group（experimental group 6）,Spred-1+/+compound Qingdai capsule group（control group 7）and Spred-1+/+model group（model group 8）were intraperitoneally injected with tamoxifen（dose 120mg/kg/mouse）once every other day for five times.On the 11th day of the experiment（1 day before modeling）,the back of mice was depilated.On the 12th day of the experiment（the first day of modeling）,the mice in the four groups were used to make models which regularly coated with imiquimod cream every day and subcutaneous injection of recombinant mouse interleukin-12（rm IL-12）and lipopolysaccharide（once a week）;At the same time,starting from the17th day of the experiment（the fifth day of modeling）,the control group5 and the experimental group 6 were given qingxuedu granule mixture,the control group 7 was given compound Qingdai capsule,and the model group8 was given normal saline.After 14 days,they were killed and sampled.In addition,the PASI score of mice was observed during the experiment;After the experiment,the skin histopathology of mice was observed,and the Western blot and RT-PCR experiments were carried out to observe the expression of related proteins and m RNA on Spred-1/Ras/ERK pathway in the skin lesions of psoriasis like mice.[Result]1.First obtain Flox heterozygous mice of spred-1 gene and Krt14-（Cre ERT2）heterozygous mice;Finally,24 spred-1 gene Flox positive and cre positive mice and 40 spred-1 gene Flox positive and cre negative mice were obtained.2.By using the modified method to construct the psoriasis like mouse model,it was found that the PASI score,pathological Baker score and spleen index of the psoriasis like mouse model after modeling were statistically significant compared with the normal group（P<0.001）,indicating that the psoriasis like mouse model induced by the modified method is more successful and can achieve the 14 day modeling effect of psoriasis like mouse model.3.In the spred-1 gene knockdown experiment,through Western blot and RT-PCR experiments,we found that the expression of spred-1 protein and m RNA in non/near skin lesions and skin lesions in the experimental 4 groups decreased significantly compared with the control 1,control 2 and control3 groups（P<0.001）,indicating that tamoxifen successfully induced and constructed spred-1 gene knockdown mice;There was no significant difference among control 1,control 2 and control 3 groups（P>0.05）,and after tamoxifen injection into non inducible conditional knockdown mice,the expression of spred-1 protein and m RNA in non/near lesions and lesions did not decrease,indicating that tamoxifen had no effect on the expression of spred-1 in non inducible conditional knockdown mice.4.In the spred-1 gene knockdown experiment,the mice in control 1,control 2,control 3 and Experiment 4 could see light erythema and thin scales from 2-3 days after modeling;From the 4th day,red patches and scales became layered and gradually aggravated;From the 9th day,the skin infiltration and thickening were obvious,and the skin hypertrophy reached the peak on the 14th day,especially in the experimental group4,which was consistent with the performance of the modified psoriasis model.In comparison,there was no significant difference in the severity of skin lesions and the change time of rash among control 1,control 2and control 3,indicating that intraperitoneal injection of tamoxifen had no intervention effect on the skin lesions of psoriasis like mice;The skin lesions in Experiment 4 were more severe than those in control 1,control2 and control 3,indicating that spred-1 knockdown could aggravate psoriasis like skin lesions in mice to a certain extent;This may be related to the down regulation of spred-1 gene in spred-1 knockdown mice induced by tamoxifen injection.Compared with control 1,control 2 and control 3,the PASI score was not statistically significant（P>0.05）,and the baker score of skin pathology was not statistically significant（P>0.05）,indicating that tamoxifen injection had no effect on the skin lesions and skin lesions pathology of modified induced psoriasis like mice.5.In the spred-1 gene knockdown experiment,the expression of ERK 1m RNA,ERK 2 m RNA,RAS m RNA,raf1 m RNA,VEGF m RNA and the protein expression of p-ERK,p-ras,RAS and VEGF in the experimental 4 groups were significantly higher than those in the control 1,control 2 and control3 groups（P<0.001）,the expression of ERK,p-raf1 protein（P<0.01）and raf1 protein（P<0.05）were also increased,which showed that spred-1gene knockdown could not inhibit the expression of Ras/ERK pathway related protein and m RNA,which exacerbated the symptoms of psoriasis like mice;Compared with control 1,control 2 and control 3 groups,the m RNA expression of ERK1,ERK 2,RAS,RAF 1,VEGF and the protein expression of p-ERK,ERK,p-ras,RAS,p-raf1,raf1 and VEGF in the three groups were not statistically significant（P>0.05）,indicating that tamoxifen had no effect on the expression of related proteins and m RNA in Ras/ERK pathway.In this experiment,the induced knockdown of spred-1 gene was mainly used,which had no effect on the disease itself.6.In the intervention experiment of traditional Chinese medicine,the expression of spred-1 m RNA in control group 5 mice was normal one day before the experiment,and there was no significant difference compared with experimental group 6,control group 7 and model group 8（P>0.05）,indicating that spred-1 gene can normally express spred-1 m RNA in four groups of mice.On the 10th day of the experiment,the expression of spred-1m RNA in control group 5 mice was normal,and the expression of spred-1m RNA in experimental group 6,control group 7 and model group 8 mice decreased significantly.There was significant difference between the two groups（P<0.001）,indicating that tamoxifen did not knock down the spred-1gene in control group 5 mice,but knocked down the spred-1 gene in experimental group 6,control group 7 and model group 8.At the same time,on the 24th day of the experiment,compared with the control group 5,the expression of spred-1 m RNA in the experimental group 6,the control group7 and the model group 8 decreased significantly（P<0.001）,indicating that the mice in the control group 5 were in the normal expression state of spred-1 gene,and the mice in the experimental group 6,the control group7 and the model group 8 were in the knockdown state of spred-1 gene;Compared with experimental group 6,the expression of spred-1 m RNA in control group 7 was lower（P<0.05）,and the expression of spred-1 m RNA in model group 8 was also lower（P<0.01）,both of which had statistical significance.It was suggested qingxuedu granule mixture up-regulated the m RNA expression of spred-1 after intragastric administration.In addition,there was no significant difference between the control group 7 and the model group 8（P>0.05）,indicating that compound Qingdai capsule had no significant effect on the m RNA expression of spred-1.7.In the intervention experiment of traditional Chinese medicine,light erythema and thin scales gradually appeared in the four groups of mice from the second and third days of modeling,and obvious red spots and scales formed layers on the fourth and fifth days.In model 8 group,with the passage of time,the color of patches gradually deepened,dark red patches appeared,scales accumulated into layers,and the skin was infiltrated and thickened,indicating that the model was successfully established by using the modified induced psoriasis like mice.In contrast,the symptoms of erythema,infiltration hypertrophy and scale in control group 5,experimental group 6 and control group 7 were significantly lighter than those in model group 8,indicating that qingxuedu granule mixture and compound Qingdai capsule have therapeutic effects on modified induced psoriasis like mice.In addition,compared with experimental group6,we found that the symptom regression rate of control group 5 was significantly faster than that of experimental group 6,indicating that qingxuedu granule mixture intervened in the treatment of psoriasis by up-regulating the expression of spred-1 protein.At the same time,there was no significant difference in the rate of erythema,infiltration and scale regression between the experimental group 6 and the control group7,indicating that after the spred-1 gene was knocked down,qingxuedu granule mixture and compound Qingdai capsule could not intervene the spred-1 protein target to inhibit the occurrence of psoriasis.8.After intragastric administration,the PASI score and skin histopathological Baker score of control group 5,experimental group 6and control group 7 were lower than those of model group 8（P<0.05）,indicating that qingxuedu granule mixture and compound Qingdai capsule can significantly improve the skin lesions and histopathological severity of modified induced psoriasis like mice.At the same time,the PASI score and Baker score of mice in control group 5 were significantly lower than those in experimental group 6 and control group 7（P<0.05）,indicating that qingxuedu granule mixture significantly increased spred-1 protein in mice without knockdown of spred-1 gene,and then reduced the skin lesion symptoms and histopathological severity of psoriasis like mice.There was no significant difference between the experimental group 6 and the control group 7 in the regression rate of erythema,infiltration and scales and the baker score of skin lesion histopathology（P>0.05）,indicating that after the spred-1 gene was knocked down,qingxuedu granule mixture could not significantly up-regulate spred-1 protein to intervene in the treatment of psoriasis,which further proved that spred-1 protein was the target of qingxuedu granule mixture in the treatment of psoriasis.9.In the intervention experiment of traditional Chinese medicine,compared with control 5,experiment 6 and control 7,the m RNA expression of ERK1,ERK2 and RAS and the protein expression of ERK,p-ras,RAS,raf1and VEGF in model 8 group were higher（P<0.001）,and the m RNA expression of raf1 and VEGF and the protein expression of p-ERK and p-raf1 were also higher（P<0.01）,indicating that after intragastric administration of qingxuedu granule mixture and compound Qingdai capsule,significantly down-regulated the expression of related proteins and m RNA in Ras/ERK pathway,and then intervened in the treatment of psoriasis.Compared with experimental group 6 and control group 7,the m RNA expression of RAS,raf1（P<0.001）,ERK2（P<0.01）,ERK1 and VEGF（P<0.05）and the protein expression of RAS,VEGF（P<0.001）,p-ras,raf1（P<0.01）,p-ERK,ERK and p-raf1（P<0.05）in psoriasis like mice decreased more significantly in control group 5,indicating that after spred-1 gene was knocked down,it can not inhibit the expression of related m RNA and protein on Ras/ERK pathway to treat psoriasis.In addition,there was no significant difference between experimental group 6 and control group 7（P>0.05）,which further showed that there was no significant difference between qingxuedu granule mixture and compound Qingdai capsule in inhibiting the expression of related m RNA and protein in Ras/ERK pathway after spred-1gene was knocked down.[Conclusion]1.Spred-1 knockdown mice were constructed by Cre-lox P system.24 non inducible spred-1 knockdown mice and 40 inducible spred-1 knockdown mice were obtained by natural reproduction.2.By evaluating the construction method of psoriasis mouse model,it is found that the improved modeling method is obviously effective,which shows that the 14 day modeling effect of psoriasis can be achieved by using the improved method.3.In the skin tissue of psoriasis like mice,the expression of Ras/ERK pathway related protein and m RNA increased after spred-1 gene was knocked down;It shows that the knockdown of spred-1 gene can not inhibit the expression of Ras/ERK pathway related proteins and m RNA,which aggravates the condition of psoriasis in mice.4.When spred-1 gene was knocked down,there was no significant difference in efficacy between qingxuedu granule mixture and compound Qingdai capsule;Qingxuedu granule mixture was used to treat psoriasis like mice without and after spred-1 gene knockdown,and there was a significant difference between the two;It fully shows that qingxuedu granule mixture can treat psoriasis by intervening spred-1 protein to regulate the expression of related proteins and m RNA in Ras/ERK pathway. |
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