The Regulating Effect Of Probiotics VSL#3 On The TLR4-NF-?B Pathway Of DSS Rats Colitis

Objective:To verify the immunoregulation of probiotics VSL#3 on TLR4-NF-?B pathway in cells and animals so as to provide new targets and ideas for the treatment of ulcerative colitis.Methods:1.Constructing a DSS-induced rats colitis model,and after the success of the model,probiotics VSL#3 was used to treat the colitis rats induced by DSS:? Group:33 Sprague-Dawly rats(SD rats)were randomly divided into 3 groups.There were 3 for blank control group,DSS model group and treatment group of probiotics VSL#3 each for 15 rats? Study object:SD rats? Testing content and methods:qPCR was used to detect the levels of TLR4,NF-?B,IL-1? and TNF-a mRNA in rats colon mucosa;Western-blot was used to detect the protein level ofTLR4?NF-?B in colon mucosa;Serum IL-1? and TNF-a in rats were detected by ELISA.2.Co-incubation of probiotics and colon cancer cells HT-29 and endotoxin LPS:? Group:blank control group(group A):colon cancer cells HT-29;model group(group B):HT-29+LPS,probiotics treatment group(group C):HT-29+LPS+VSL#3? Study object:colon cancer cells HT-29?Testing content and methods:Total RNA and total protein were extracted,levels of TLR4.NF-?B?IL-1?and TNF-a mRNA were detected by qPCR.The protein expression of TLR-4?NF-?B?IL-1? and TNF-awere detected by Western-blot.3.Statistical processing:Adopting IBM SPSS 24 statistical software package to analyze data.Results:1.Animal experiments:(1)After making the model,the rat DAI score of the model group reached the score of 9.4 ± 0.48,and the pathological score was 7.8 ± 1.04,which was significantly higher than that of the blank control group(P<0.05).(2)In the probiotics treatment group,DAI integral gradually reduced with the extension of treatment time.When finishing treatment,the DAI score of the probiotics treatment group was significantly lower than that in the DSS model group(P<0.05).(3)The lesion degree of colonic inflammation in the probiotics group significantly reduced compared with that of DSS model group,and the colon histopathological score of the rate was lower than that of the DSS model group(P<0.05).(4)The expression of TLR4.NF-?B?TNF-??IL-1?mRNA in the rat colon tissue:compared with the blank control group,the expression of TLR4?MyD88?NF-?B?TNF-??IL-1?mRNA in the rat colon tissue of DSS model group significantly enhanced(P<0.01).After treatment with probiotics VSL#3,compared with the DSS model group,the expression of TLR4?NF-KB?TNF-??IL-1 PmRNA in rats colon tissue of probiotics treatment group significantly reduced(P<0.05).(5)The expression of TLR4?NF-?B protein in the rat colon tissue:compared with the blank control group,the expression of TLR4?NF-?B protein in the rat colon tissue of DSS model group significantly enhanced(P<0.01).After treatment with probiotics VSL#3,compared with DSS model group,the expression of TLR4?NF-?B protein in the rat colon tissue of probiotics treatment group decreased significantly(P<0.01).(6)The expression level of serum IL-1?and TNF-a:compared with the blank control group,the expression of serum serum IL-1?and TNF-?in DSS model group significantly increased(P<0.01).After treatment with probiotics VSL#3,compared with the DSS model group,the expression of serum IL-1?and TNF-ain probiotics treatment group significantly reduced(P<0.001).2.Cell experiments:(1)The expression of TLR4?NF-kB?TNF-??IL-1?mRNA in HT-29 cells:the expression of TLR4.NF-?B.TNF-?.IL-1?mRNA in the model group is highest:TLR4,and their relative expressions were 5.78± 1.11.2.03±0.16.2.81±0.38?1.0910.02.After a week of incubation with probiotics,compared with the model group,the expression of TLR4?NF-?B?TNF-??IL-1?mRNA in probiotics treatment group decreased,which had statistically significance(P<0.05).(2)The expression of TLR4?NF-?B?TNF-??IL-1? protein in HT-29 cells:the expression of TLR4?NF-?B?TNF-??IL-1? protein in the model group is the highest,and their relative expressions were 1.24 ±0.18.1.13 ±0.18.0.6±0.09.1.08 ±0.1.After a week of incubation with probiotics,the expression of TLR4?NF-?B?TNF-??IL-1? protein all decreased in different level,but compared with the model group,the difference didn't have statistically significance(P>0.05).Conclusions:1.The model of ulcerative colitis of SD rats can be successfully induced by DSS model.After Ht-29 cells were incubated for 12h with Human TNF-a(20mg/ml,and then for 15 hours with LPS(1ug/ml),the inflammatory cell model was successfully constructed.2.Probiotics VSL#3 can improve the clinical symptoms of UC rats and the degree of inflammation in the colon tissue.3.In the animal experiments and cells(in vivo and in vitro)experiments,probiotics VSL#3 can lower the expression of the upstream and downstream factor of TLR4-NF-?B pathway in mRNA and protein level.And it has different degree of intervention and regulation function for TLR4.NF-?B TNF-??IL-1?.Probiotics VSL#3 may achieve the goal of treating ulcerative colitis by inhibiting the TLR4-NF-?B pathway so as to inhibit immune and suppress inflammatory response.