Effect Of Thalidomide On NF-κB Signaling Pathway In Mice With Systemic Sclerosis Induced By Bleomycin

Objective: In this paper,we established a mouse model of systemic sclerosis(SSc)induced by bleomycin(BLM)to observe the effect of different doses of thalidomide(Thal)on VEGF mRNA in SSc mouse model.Methods: 40 female BALB/c mice aged 6-8 weeks were selected,and SSc mouse models were established by subcutaneous injection of BLM.The Thal(10)group,Thal(20)group,and Thal(30)group were given 10 mg respectively./kg/d,20mg/kg/d,30mg/kg/d Thal solution was given by gavage,the con group and SSc group were given gavage of the same volume of distilled water,and the mice were sacrificed on the 28 th day of gavage treatment And lungs.HE and Masson’s staining were performed to observe the thickness and inflammation of mouse skin tissue and the degree of inflammation and fibrosis of lung tissue under microscope;the hydroxyproline(HYP)of mouse skin and lung tissue was detected by sample alkaline hydrolysis method Content;RT-PCR method was used to detect the expression of vascular endothelial growth factor(VEGF)mRNA in the skin and lung tissues of mice in each group.Results:(1)Compared with con group,SSc group had increased skin thickness and inflammation score(all P<0.01),lung tissue inflammation score and fibrosis increased(all P <0.01);compared with SSc group,Thal(10)Group,Thal(20)group,Thal(30)group,skin thickness and inflammation,lung tissue inflammation score,fibrosis were reduced to varying degrees,skin thickness F value = 24.141,P <0.01.Skin inflammation score F value=81.305,P <0.01,lung tissue inflammation score F value=76.211,P<0.01.F value of pulmonary fibrosis=103.832,P <0.01.(2)Detection of HYP content showed that: compared with the con group,the HYP in the skin and lung tissues of the SSc group increased(both P <0.05);compared with the SSc group,the Thal(10)group and Thal(20)The HYP content in the skin and lung tissues of mice in group and Thal(30)group decreased(P <0.05),and the degree of decrease was proportional to the concentration of administration.(3)RT-PCR results showed that compared with the con group,the VEGF mRNA expression in the skin and lung tissues of the SSc group increased(both P <0.01);compared with the SSc group,the lung tissue Thal(10)group was not significant Difference(P >0.05),the expression of VEGF mRNA in lung tissues of Thal(20)group and Thal(30)group decreased(P <0.05,P <0.01).Compared with SSc group,the expression of VEGF mRNA in the skin of Thal(10)group,Thal(20)group and Thal(30)group decreased(P <0.05,P<0.01,P<0.01)Conclusion: Thal can improve the skin thickness and inflammation of the SSc mouse model,as well as the degree of inflammation and fibrosis of the lung tissue,reduce the content of hydroxyproline,and inhibit the expression of VEGF mRNA.Objective: To establish a bleomycin(BLM)-induced systemic sclerosis(SSc)mouse model and observe the mechanism of different doses of thalidomide(Thal)on the NF-κB signaling pathway of SSc mouse model.Treatment of SSc provides a theoretical basis.Methods: 40 female BALB/c mice aged 6-8 weeks were selected,and SSc mouse models were established by subcutaneous injection of BLM.The Thal(10)group,Thal(20)group,and Thal(30)group were given 10 mg respectively./kg/d,20mg/kg/d,30mg/kg/d Thal solution was given by gavage,the con group and SSc group were given gavage of the same volume of distilled water,and the mice were sacrificed on the 28 th day of gavage treatment And lungs.HE and Masson’s staining were performed to observe the thickness and inflammation of mouse skin tissue and the degree of inflammation and fibrosis of lung tissue under microscope;the hydroxyproline(HYP)of mouse skin and lung tissue was detected by sample alkaline hydrolysis method Content;RT-PCR method was used to detect nuclear transcription factor-κB(NF-κB,P65),tumor necrosis factor α(TNF-α),interleukin-1β(IL-1β),interleukin in the skin and lung tissues of mice in each group-6(IL-6)mRNA expression level;Western blot(WB) detection of NF-κB(P65),phosphorylated NF-κB(p-P65),TNF-α,IL-6 protein expression.result:(1)RT-PCR results of mouse skin tissue showed that compared with con group,SSc group NF-κB P65,IL-1β,IL-6,TNF-α mRNA expression increased(all P <0.01);and SSc Compared with the Thal(10)group,there was no statistically significant difference in the expression of NF-κB P65 mRNA,and the expression of NF-κB P65 mRNA in the Thal(20)and Thal(30)groups decreased(both P <0.01).Compared with the SSc group,there was no statistically significant difference in the expression of IL-1β and IL-6 mRNA in the Thal(10)group,while the expression of IL-1β and IL-6 mRNA in the Thal(20)and Thal(30)groups decreased.(P <0.05,P <0.01).Compared with the SSc group,there was no statistically significant difference in the expression of TNF-α mRNA in the Thal(10)group.The expression of TNF-α mRNA in the Thal(20)and Thal(30)groups decreased(both P <0.05).(2)RT-PCR results of mouse lung tissue showed that compared with con group,SSc group NF-κB P65,IL-1β,IL-6,TNF-α mRNA expression increased(all P <0.01);and SSc Compared with the Thal(10)group,there was no statistically significant difference in the expression of NF-κB P65 and IL-6(P>0.05).The expression levels of NF-κB P65 and IL-6 in the Thal(20)group and Thal(30)group Decrease(P <0.05,P <0.01).Compared with the SSc group,there was no statistically significant difference in the expression of IL-1β mRNA in Thal(10)group(P >0.05).The expression of IL-1β mRNA in Thal(20)group and Thal(30)group decreased(both P <0.01).Compared with SSc group,Thal(10)and Thal(20)groups had no statistically significant difference in TNF-α mRNA expression(P >0.05),Thal(30)group TNF-α mRNA expression decreased(P <0.01).(3)Western blot results of mouse skin tissue showed that compared with the con group,the SSc group IL-6,NF-κB P65,p-P65,TNF-α protein expression was significantly increased(P <0.01,P <0.05,P <0.01,P <0.01),compared with the SSc group,Thal(10)group IL6 protein expression was not statistically different(P >0.05),Thal(20)group,Thal(30)group IL6 protein expression decreased(All P <0.05).Compared with SSc group,Thal(10)group and Thal(20)group NF-κB P65,p-P65,TNF-α protein expression levels were not statistically different(P >0.05),Thal(30)group NF-κB P65,p-P65 and TNF-α protein expression decreased(all P <0.05).(4)Western blot results of mouse lung tissue showed that compared with the con group,the SSc group IL-6,NF-κB P65,p-P65,TNF-α protein expression was significantly increased(P <0.01),compared with the SSc group Compared with the Thal(10)group and Thal(20)group,there was no statistically significant difference in the expression of IL-6 and TNF-α protein(all P >0.05),while the Thal(30)group had the same expression of IL-6 and TNF-α protein Decrease(P<0.05).Compared with the SSc group,there was no statistically significant difference in the expression of NF-κB P65 protein in the Thal(10)group(P >0.05).The expression of NF-κB P65 protein in the Thal(20)group and Thal(30)group decreased(P < 0.05,P <0.01).Compared with the SSc group,the p-P65 protein expression in the Thal(10)group,Thal(20)group,and Thal(30)group was significantly reduced(all P <0.01).Conclusion: Thalk may block the inflammatory response of SSc by inhibiting the activation of NF-κB signaling pathway and the expression levels of cytokines IL-1β,IL-6,and TNF-α.