| Obesity and obesity-related diseases,such as type II diabetes,cardiovascular and cerebrovascular diseases,cancer has become a worldwide concern [1].Metabolic disorders is the most fundamental reason in the process of obesity,and metabolic syndrome is the most basic link of lipid metabolism disorders [2].Lipid metabolic disorder is due to internal and external environmental factors caused the expre ssion of lipid metabolites and metabolic abnormal distribution [3].A large number of studies have confirmed that the blood free fatty acid expression is significantly increased in the process of lipid metabolism disorders.(FAT)/CD36 as one of the importa nt metabolic enzymes of fatty acids in a variety of tissue cells have a wide range of distribution,Detection of FAT/CD36 expression can better observe the body's inflammatory response to the molecular regulation process.The expression of FAT/CD36 can be detected in many obesity and inflammatory models.However,the mechanism of FAT/CD36 expression changes is not clear [4].In the inflammatory response,the inflammatory immune factor PTX3 is an important component of the body's innate immune body fluid com position,mainly through the interaction with the C1 q and H factors,activation and regulation of complement cascade reaction,play an immune regulatory role in resistance to specificity microorganisms and play an important role in the regulation of inflam mation [5].In recent years,the relationship between PTX3 and obesity and inflammation has become more and more in-depth researches,obesity is a low abundance chronic inflammation has become a recognized fact [6].However,whether inflammation can directly caused obesity is rarely reported,a large number of experiments have shown that many chronic diseases can be traced back to the early life,Intrauterine inflammation causes offspring metabolic diseases and obesity as an independent factor in vital cong enital obesity [7];In the past,most of the inflammation and obesity models are through the mouse acquired inflammatory stimulation or diet induced,it is more difficult to reflect the true situation of animal inflammation due to human intervention factors,so our model selected the inflammatory stimuli from the pregnancy mice to study the growth and metabolic status of the offspring mice and to study the relationship between obesity and inflammation.It was more natural and appropriate to response the bod y of the real situation of inflammation because of the absence of any human intervention to offspring mice,through our pre-study has been found that disposable pregnancy inflammatory stimulation can lead to obesity in offspring mice [7,8],but its specific mechanism is not clear,so this paper from fat metabolism Regulatory factor FAT/CD36 and inflammatory immune factor PTX3 point of view,to explore the pathways between inflammation and obesity regulation mechanism,Further studies of the relationship be tween disease and obesity.Methods1.Effects of pregnancy inflammation on Fat Development,Lipid Metabolism and expression of FAT/CD36 in Offspring Mice1.1 The C57 mice purchased from the Third Military Medical University were Feeding in the SPF environment for 8 weeks of age,the female body weight is(20±2)g and male mice(25 ± 2)g.According to the female: male = 2: 1 ratio of the same cage overnight,Separate of cage feeding in the second day,recorded as pregnant 0 days,on the eleventh day weighing the weight of pregnant rats if the weight was significantly greater than 2g and observe the mouse abdominal obvious uplift,then determine the pregnancy.1.2 pregnant mice were randomly divided into two groups,the control group NS: in the pregnancy 11 days disposable intraperitoneal injection of sterile saline 0.2ml;LPS group: disposable intraperitoneal injection of LPS 75?g.kg-1,taking the offspring mice as the research objects after pregnancy,build the model of the pregnancy inflammation.1.3 Two groups of offspring mice were monitored for body weight from birth(set to day 0),every once 2 weeks until 12 weeks.Respectively,Weigh the fat weight(g)and the fat coefficient(fat coefficient = fat weight/body weight × 100)was calculated in 4 w,8 w and 12 w.1.4 Offspring male mice eyeball blood collection in 4 weeks,8w and 12 w.The levels of free fatty acids(FFA)and triglyceride(TG)in the blood of male mice were detected by means of kits.1.5 Offspring male mice take epididymal adipose tissue.The contents of free fatty acids(FFA)and triglyceride(TG)were measured in the male adipose tissue by means of kits.1.6 Real-time PCR was used to detect the expression of FAT/CD36 m RNA in epididymal fat of male offspring.Western blot was used to detect the expression of FAT/CD36 protein in adipose tissue of male offspring.1.7 Real-time PCR and Western Blot were used to detect the expression of FAT/CD36 in the induced differentiation 3T3-L1 cells.2.Study the mechanism of inflammation stimulation during pre gnancy effect on offspring mice2.1 The expression of a P2,PPAR?,CEBP/?,CEBP/? m RNA and protein were detected by Real-time PCR and Western Blot in the mice epididymal dipose tissue.2.2 The expression levels of PTX3 m RNA and protein were detected by Real-time PCR and Western Blot in the 4W,8W and 12 W in mice epididymal adipose tissue.2.3 RT-PCR and western blotting analysis of PTX3 and adipocyte differentiation markers(aP2?PPAR??CEBP/??CEBP/?)in the 3T3-L1 that were transfected with si RNA and overexpressed PTX3 plasmid.2.4 Flow cytometry detection of transfected si RNA and overexpression of PTX3 plasmid on 3T3-L1 cell apoptosis2.5 MTT assay was used to detect the effect of si RNA and overexpression of PTX3 on the proliferation of 3T3-L1 cells2.6 The effects of transfection of si RNA and overexpression of PTX3 on the differentiation of 3T3-L1 cells were investigated by oil red staining.2.Maternal Pregnancy Inflammation Effects on Offspring Mice Fat Development2.1 The expression levels of PTX3 m RNA and protein were detected by Real-time PCR and Western Blot in the 4W,8W and 12 W Epididymal Adipose Tissue.2.2 Real-time PCR and Western Blot were used to detect the m RNA and protein levels of adipose differentiation markers(a P2,PPAR?,CEBP/?,CEBP/?)in the epididymal fat of male offspring.2.3 Effects of transfection of si RNA and overexpression of PTX3 on m RNA expression of PTX3,adipogenic differentiation markers(aP2,PPAR?,CEBP/?,CEBP/?)and adipogenic differentiation protein in 3T3-L1 cells.2.4 Flow cytometry detection of transfected si RNA and overexpression of PTX3 plasmid on 3T3-L1 cell apoptosis2.5 MTT assay was used to detect the effect of si RNA and overexpression of PTX3 on the proliferation of 3T3-L1 cells2.6 The effects of transfection of si RNA and overexpression of PTX3 on the differentiation of 3T3-L1 cells were investigated by oil red staining.2.7 Western Blot was used to detect the relative expression of MAPK pathway-associated protein in adipose tissue of male offspring.Results1.Maternal Pregnancy Inflammation Effects on Offspring Mice Fat Development1.1 Compared with NS group,the body weight,epididymal fat weight and 4W,8W epididymal fat coefficient of LPS group were significantly higher than those of NS group(p<0.05)(Fig.1-3);8W,12 W offspring female body weight,4W,8W,12 W offspring female perinatal fat wet weight and 8W,12 W perinatal fat coefficient were significantly increased,this differences were statistically significant(p<0.05)(Fig.1-3).1.2 Compared with NS group,the expression of FFA in blood of 4w,8w and 12 w offspring of LPS group was significantly higher than that of NS group(p<0.05)(Fig.4A);The content of TG in 8w and 12 w of blood in LPS group was significantly higher than that in control group(p<0.05)(Fig.4B).1.3 Compared with NS group,the content of FFA in adipose tissue of 4w,8w and 12 w offspring of LPS group was significantly increased(P<0.05)(Fig.5A);In the LPS group,the content of TG in the adipose tissue of the offspring male was significan tly increased at 8w and 12 w,the difference was statistically significant(p<0.05)(Fig.5B).1.4 Compared with NS group,the expression of FAT/CD36 m RNA in the epididymis adipose tissue of male of LPS group was significantly increased(p<0.05)(Fig.6);T he expression of FAT/CD36 protein in the epididymis adipose tissue of male mice in LPS group was significantly increased at 4w and 8w(p<0.05)(Fig.7)1.5 The expression of FAT/CD36 m RNA in 3T3-L1 cells was significantly higher than that in the normal group(p<0.01)(Fig.8);The expression level of FAT/CD36 protein in 3T3-L1 cells was significantly increased(P<0.01)(Fig.9).2.Study the mechanism of inflammation stimulation during pregnancy effect on offspring mice2.1 Compared with NS group,the expression of PTX3 m RNA and protein in LPS group were significantly increased(p<0.05)(Fig.1 0).2.2 Compared with NS group,m RNA expression and protein level of adipogenic differentiation markers were significantly increased in offspring mice 4W CEBP/?,CEBP/?,PPAR? and 8W CEBP/?,PPAR? and 12 W CEBP/?,a P2 and PPAR? High(p<0.05)(Fig.11)2.3 The expression of PTX3 and adipogenic differentiation markers(CEBP/?,aP2)was significantly lower than that of the control group(p<0.05)(Fig.12),and the difference was statistically significant(p<0.05);Compared with the control group,the overexpression PTX3 plasmid group adipogenic differentiation markers(aP2,PPAR?,CEBP/?,CEBP/?)in 3T3-L1 was significantly increased(p<0.05)(Fig.12).2.4 Compared with the control group,the overexpression group had no significant effect on the apoptosis of 3T3-L1 cells,and the inhibition expression PTX3 significantly reduced the apoptosis of 3T3-L1 cells(p<0.05)(Fig.13A).2.5 Compared with the control group,the overexpression group and the inhibitory expression group had no significant effect on the cell cycle of 3T3-L1(p>0.05)(Fig.13B).2.6 The results of oil red staining showed that the degree and number of mature cells were significantly increased in the overexpressi on of PTX3 group,and the differentiation degree and number of cells in the inhibitory expression group were significantly decreased(p<0.05)(Fig.13C)2.7 Compared with the control group,the overexpression group and the inhibitory expression group had no significant effect on the proliferation of 3T3-L1 cells(p>0.05)(Fig.13D).2.8 Compared with NS group,non-phosphorylated MAPK(P38,JNK,ERK1/2)related protein 4W ERK1/2(P42/P44)and JNK/SNPK(P46 P54),8W ERK1/2(P42/P44)and 12 W JNK/SNPK(P46/P54)in adipose tissue of LPS group were significantly decreased,the difference was statistically significant(p<0.05)(Fig.11A-C);Phosphorylated MAPK(p-P38,p-JNK/SNPK,p-ERK1/2)-related protein p-P38,p-JNK/SNPK(p-P42/ P44),p-ERK1/2(p-P46/P54)Was significantly increased(p<0.05)(Fig.14D-F).Conclusions1.Maternal inflammation during pregnancy,offspring mice appeared to increase the weight,fat weight,fat coefficient and the expression of FFA and TG in the blood and adipose tissue,Which are lipid metabolism disorders,The expression of FAT/CD36 in the 3T3-L1 cells was also significantly higher than that in the normal group,indicating that FAT / CD36 plays an important role in the process of lipid metabolism disorder in the offspring caused by inflammation during pregnancy.2.Maternal inflammation LPS stimulation during pregnancy were established inflammation and obesity models that showed a significant increase in PTX3 levels in offspring mice.Highly expressed PTX3 promotes the formation of adipocytes by positively regulating the expression of adipose differentiation markers,leading to obesity.This effect of PTX3 is most likely to be regulated by the MAPK pathway,So PTX3 in the process of obesity has played a negative role in increasing th e susceptibility of obesity. |
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