Metabonomics Study Of Safflower Against Skin Fibrosis In Mice With Systemic Sclerosis

ObjectivesTo bserve the effect of safflower(HH)on skin tissue fibrosis of bleo mycin-induced systemic sclerosis(SSc)mice,and perform metabonomics t echnology on the skin of SSc mice based on UHPLC-Q-TOF/MS Metabo nomics analysis to explore the mechanism of safflower anti-skin fibrosis i n SSc mice.Method40 SPF female Balb/c mice were divided into a blank control group(Control group),scleroderma model group(SSc group),prednisone acetate group(SSc+Pred group),and safflower according to the random number table method.In the water extract group(SSc+HH group),10 mice in each group were induced by bleomycin to establish a systemic sclerosis(SSc)mouse model.Except for mice in the Control group,mice in the control group were injected with a concentration of 0.1 mol/l PBS solution(p H=7.2)subcutaneously on their backs,and mice in the other groups were injected with a concentration of 500μg/ml BLM buffer subcutaneously on their backs.The mice in each group were injected with 0.1ml/d according to the above method,once a day for 28 days.At the same time of modeling,control group and SSc group were given 0.2ml/d normal saline by gavage,SSc+HH group was given safflower water extract0.2ml/d,SSc+Pred group was given prednisone acetate solution 0.2ml/d Gavage.Observe the general state of each group of mice(mental state,diet,activity,sensitivity,back hair gloss and growth rate,skin softness and hardness,etc.)and weight changes.After modeling and gavage for 28 consecutive days,the mice of each group were sacrificed.The skin tissues of the mice were taken,and the pathological changes of the skin tissues of the mice of each group were observed by HE staining and Masson staining,and the changes of dermal thickness and tissue collagen fiber content were detected;The metabonomics technology of UHPLC-Q-TOF/MS is used to detect the changes of metabolites in the skin tissues of mice in each group,and the changes of the metabolites in the skin tissues of each group of mice are detected by principal component analysis(PCA)and orthogonal partial least squares.Multiplying discriminant analysis(OPLS-DA)and other multivariate statistical analysis to screen out the endogenous biomarkers in the skin tissues of each group of mice,and then obtain the related metabolic pathways involved,and find that the differential metabolites and safflower have a differential metabolism To further explore the mechanism of safflower against tissue fibrosis in SSc mice.Results1.General conditionsControl group mice have good mental state,normal diet,lively and active,clean and shiny back hair,rapid growth,and soft skin;SSc group mice have poor mental state,reduced diet,prefer static and evil movements,slow response,and model back There was almost no hair growth in the area,and the skin became hard and thickened.Compared with the SSc group,mice in the SSc+Pred group showed better attitude and diet,with better mobility,gradually increasing back hair,normal gloss,and softer skin.The mental state of the mice in the SSc+HH group was close to normal,with good mobility,hair growth on the back,and skin softness close to normal.2.The change of weightThe body weights of control group was increasely.Compared with the control group,body weights that obviously decreased in model group(p<0.01),After 28 days of treatment with safflower,and the body weights increased significantly(p<0.01).3.HE staining of skin tissue and changes in dermis thicknessThere were no obvious pathological changes in the skin tissues of the Control group mice,the morphology and structure were complete,the hair follicles were visible under the dermis,and there was no obvious inflammatory cell infiltration;Compared with the Control group,the SSc group mice skin epidermis spinous layer was hypertrophy,the dermis layer was obviously thickened,and the skin The accessory fat layer and muscle layer were atrophied,inflammatory cell infiltration was seen around,and collagen fibers were not significantly proliferated;compared with the SSc group,the number of fibroblasts and red collagen fibers in the skin tissue of the SSc+HH group decreased,and the morphology tended to be Normal,there is no obvious disorder in the arrangement.Both the SSc+HH group and the SSc+Pred group showed thinning of the dermis,improved proliferation of collagen fibers,and increased subcutaneous appendages.4.Masson staining of skin tissue and changes in collagen fiber contentThe collagen fiber content in the skin tissue of mice in the Control group did not change significantly;compared with the Control group,the collagen fiber content in the skin tissue of mice in the SSc group was significantly increased(p<0.01).The collagen fiber content in the skin tissues of mice in the SSc+Pred group and SSc+HH group was significantly lower than that in the SSc group(p<0.01).5.Screening of biomarkers for skin tissue metabolismA total of 23 endogenous biomarkers with significant differences wer e screened in the positive and negative ion modes,namely Adenosine,NOleoylethanolamine D-Pipecolinic acid,L-Asparagine,L-Methionine,L-Leu cine,Citraconic acid,Glyceric acid,Linolenic acid,Cyclohexylsulfamate,Docosahexaenoic acid,Glycerol 3-phosphate,Pantothenate,L-Threonine,D odecanoic acid,Phenylpyruvate,L-Glutamate,DL-3-Phenyllactic acid,L-Trypt ophan Acid,L-Alanine,L-Phenylalanine,L-Valine,D-Aspartic acid.6.The potential serum metabolic analysis biomarkers.6.Related metabolic pathway analysisThe screened endogenous biomarkers involved a total of 20 metabolic pathways.In this experiment,the 9 most significant pathways were selected based on the number of markers involved in the relevant pathways.Protein digestion and absorption,ABC transporters(ABC transporter),Aminoacyl-t RNA biosynthesis,Biosynthesis of amino acids,Central carbon metabolism in cancer Glycine,serine and threonine metabolism(Glycine,serine and threonine metabolism),Alanine,aspartate and glutamate metabolism,Biosynthesis of unsaturated fatty acids.2-Oxocarboxylic acid metabolism.Conclusions1.Safflower can improve the dermal thickness and fibrosis of the skin of SSc mice induced by bleomycin,and has the effect of anti-SSc mouse skin fibrosis.2.The skin metabolism profile of SSc mice has changed significantly,which obviously disturbs amino acid biosynthesis and metabolism,energy metabolism and lipid metabolism,ABCtransporter,aminoacyl-t RNA biosynthesis,and central carbon metabolism in cancer.3.HH has varying degrees of regulation on abnormally changed metabolite levels and related metabolic pathways in the skin tissues of SSc mice.It may regulate amino acid metabolism,energy metabolism and lipid metabolism,ABC transporters,and aminoacyl-t RNA organisms.Synthesis,central carbon metabolism in cancer,2-oxocarboxylic acid metabolism and anti-SSc skin fibrosis effect.