| Purpose:From the perspective of inflammation,lipopolysaccharides(LPS)was used to induce human colonic epithelial cells(HCoEpiC)to create UC cellular inflammation model.The pro-inflammatory cytokines and anti-inflammatory cytokine are tested to observe the anti-inflammatory ability of Qingdai.And by detecting the regulation of Qingdai on key molecules in the PI3K/Akt and NF-κB signaling pathway,it is clear that Qingdai can exert its anti-inflammatory effect.Material and method:Selection of the HCoEpiC culture conditions for cellular inflammation model:The frozen HCoEpiC cells were resuscitated,cultured,passaged and frozen,and kept for subsequent experiments.The HCoEpiC cells in the logarithmic growth phase were divided into 4 groups,namely the control group,5μg/mL group,10μg/mL group,and 15μg/mL group.In addition to the control group,the other three groups were added with 10μL of LPS DEME culture medium at concentrations of 5μg/mL,10μg/mL,and 15μg/mL.Use the Enzyme-linked immunoadsordent detects the content of IL-6,calculates the linear regression equation of the standard curve according to the concentration of the standard substance and the corresponding OD value,and then calculates the corresponding IL-6 concentration on the regression equation according to the OD value of the sample to determine the optimal concentration of LPS to induce inflammatory response.Then,HCoEpiC cells in logarithmic growth phase were inoculated into 96-well plates at 1.0×10~8,100μL/well,and cultured for 12h,24h,and48h respectively,and then the cell viability was detected by MTT method to determine the best culture time for cell viability.Preparation of drug-containing serum of rats:SPF-grade healthy male SD rats were divided into 5 groups according to their body weight using the random number table method:control group,SASP group,high-dose Qingdai group,medium-dose Qingdai group,and low-dose Qingdai group,each group has 6 rats.According to the human and rat dosage conversion formula,the rats in each group were given intragastrically,and the intragastric concentrations were 0.024g/mL,0.054g/mL,0.018g/mL,0.009g/mL,and the intragastric dose was 3mL/d.In the control group,the same volume of distilled water was given by gavage.After continuous gavage for 5 days,5%chloral hydrate0.7mL/100g was injected intraperitoneally for anesthesia,and blood was taken from the abdominal aorta.After the blood was taken,the serum was separated to form rat drug-containing serum,which was placed in a refrigerator at-80°C for later use.The detection of pro-inflammatory cytokines and anti-inflammatory cytokines:HCoEpiC cells in logarithmic growth phase were inoculated into 96-well plates at 1.0×10~8,100μL/well,and divided into 6 groups:control group,model group,SASP group,Qingdai high group,Qingdai middle group,Qingdai low group.The control group was added with 10%calf serum+DEME culture medium,the model group was added with10μg/mL LPS on the basis of the control group,and the SASP group was added with rat serum containing SASP on the basis of the model group.Qingdai high,medium and low group was added with rat serum containing high,medium and low doses of Qingdai on the basis of the model group.After 24 hours of culture,the ELISA method was used to detect the changes of the pro-inflammatory cytokines IL-6,TNF-α,IL-1β,IL-12 and the anti-inflammatory cytokines IL-4 and IL-10 in the serum.The effects of Qingdai on inflammatory cytokines were tested in two aspects:pro-inflammatory and anti-inflammatory.The influence of Qingdai on PI3K/Akt signaling pathway:The establishment and grouping of HCoEpiC cellular inflammation model are the same as above,the relative expression of PI3K and AktmRNA in HCoEpiC cells was detected by QPCR method,and PI3K,Akt,p-Akt protein was detected by Western Blot.The influence of Qingdai on the NF-κB signaling pathway:The establishment and grouping of HCoEpiC cellular inflammation model are the same as above.The relative expression of IKKα,IκBα,NF-κB m RNA in HCoEpiC cells was detected by QPCR,and IKKα、p-IKKα、IκBα,p-IκBα,NF-κB protein expression were detected by Western Blot.Results:Experimental results of the first paper:1.The effect of different concentrations of LPS on the inflammatory cytokine IL-6The expression of IL-6 in each group was measured by ELISA.The results showed that compared with the control group,the relative expression of IL-6 in other groups increased significantly,and the difference was statistically significant(P<0.01).Compared with the 5μg/mL group,the relative expression of IL-6 in the 10μg/mL and15μg/mL groups increased significantly,and the difference was statistically significant(P<0.01).However,there was no significantly difference in the relative expression of IL-6 between the 10μg/mL and 15μg/mL groups(P>0.05).2.The effect of different culture time on cells viabilityThe MTT method was used to detect the activity of HCoEpiC cells under different culture times.Compared with 12h group,the OD values of 24h and 48h groups increased significantly,and the difference was statistically significant(P<0.01).Compared with 24h group,although the average OD value of 48h increased,the difference was not statistically significant(P>0.05).Experimental results of the second paper:1.The effect of Qingdai on the pro-inflammatory cytokine IL-6 in HCoEpiC cellular inflammation modelThe expression of pro-inflammatory cytokine IL-6 in HCoEpiC cells induced by LPS was significantly increased,and the difference was statistically significant(P<0.01).After drug intervention,the IL-6 content of each group was significantly lower than that of the model group,and the difference was statistically significant(P<0.01).Compared with the SASP group,the IL-6 content of the high Qingdai group decreased more significantly,and the difference was statistically significant(P<0.01);the IL-6 content of the low Qingdai group increased,and the difference was statistically significant(P<0.05);There was no significant difference in IL-6 content between the middle group of Qingdai(P>0.05).Compared with the Qingdai high group,the IL-6content of the other two groups increased compared with the Qingdai high group,and the difference was statistically significant(P<0.01);while there was no statistical difference in the IL-6 content between the Qingdai middle group and the Qingdai low group(P>0.05).2.The effect of Qingdai on the pro-inflammatory cytokine TNF-αof HCoEpiC cellular inflammation modelThe expression of pro-inflammatory cytokine TNF-αin HCoEpiC cells induced by LPS increased significantly,and the difference was statistically significant(P<0.01).After drug intervention,compared with the model group,the content of TNF-αin each drug group was significantly reduced,and the difference was statistically significant(P<0.01).Compared with the SASP group,the TNF-αcontent of the Qingdai high group decreased more significantly,and the difference was statistically significant(P<0.01);the TNF-αcontent of the middle Qingdai group and the low Qingdai group increased,and the difference was statistically significant(P<0.05,P<0.01).Compared with the Qingdai high group,the TNF-αcontent of the other two groups increased and the difference was statistically significant(P<0.01);while the Qingdai middle group and the Qingdai low group had no significant difference(P>0.05).3.The effect of Qingdai on the pro-inflammatory cytokine IL-1βof HCoEpiC cellular inflammation modelThe expression of pro-inflammatory cytokine IL-1βin HCoEpiC cells induced by LPS was significantly increased,and the difference was statistically significant(P<0.01).After drug intervention,the IL-1βcontent of each group was significantly lower than that of the model group,and the difference was statistically significant(P<0.01).Compared with the SASP group,the IL-1βcontent of the Qingdai high group was significantly reduced,and the difference was statistically significant(P<0.01);the IL-1βcontent of the Qingdai low group was increased,and the difference was statistically significant(P<0.05);There was no statistically significant difference in IL-6 content in the middle group(P>0.05).Compared with the Qingdai high group,the IL-1βcontent of the other two groups increased and the difference was statistically significant(P<0.01);while there was no statistical difference in the IL-1βcontent between the Qingdai middle group and the Qingdai low group Academic significance(P>0.05).4.The effect of Qingdai on the pro-inflammatory cytokine IL-12 of HCoEpiC cellular inflammation modelLPS induced HCoEpiC cells to produce an inflammatory response,and the expression of the pro-inflammatory cytokine IL-12 was significantly increased,and the difference was statistically significant(P<0.01).After drug intervention,compared with the model group,the IL-12 content of each drug group was significantly reduced,and the difference was statistically significant(P<0.01).Compared with the SASP group,the IL-12 content of the Qingdai high group was lower,and the difference was statistically significant(P<0.01);the IL-12 content of the middle and low group was significantly increased,and the difference was statistically significant(P<0.01).Compared with the Qingdai high group,the IL-12 content of the other two groups increased,and the difference was statistically significant(P<0.01);and the Qingdai low group had a more significant increase in IL-12 content than the Qingdai middle group.The difference was statistically significant(P<0.01).5.The effect of Qingdai on the anti-inflammatory cytokine IL-4 of HCoEpiC cellular inflammation modelThe anti-inflammatory cytokines of HCoEpiC cells induced by LPS were significantly reduced compared with the normal group,and the difference was statistically significant(P<0.01).After drug intervention,compared with the model group,the IL-4 content of each drug group increased significantly,and the difference was statistically significant(P<0.01).Compared with the SASP group,the IL-4 content of each group of Qingdai increased significantly,and the difference was statistically significant(P<0.01).Among the three dose groups of Qingdai,the high Qingdai group was significantly higher than the other two groups,and the difference was statistically significant(P<0.01).The middle Qingdai group was significantly higher than the Qingdai low group and the difference was statistically significant(P<0.01).6.The effect of Qingdai on the anti-inflammatory cytokine IL-10 of HCoEpiC cellular inflammation modelThe anti-inflammatory cytokine IL-10 of HCoEpiC cells induced by LPS was significantly reduced compared with the control group,and the difference was statistically significant(P<0.01).After drug intervention,compared with the model group,the IL-10 content of each drug group increased significantly,and the difference was statistically significant(P<0.01).Compared with the SASP group,the levels of IL-10 in the high and middle groups of Qingdai increased significantly,and the difference was statistically significant(P<0.01),but there was no significant difference compared with the Qingdai low group(P>0.05).Compared with the three dose groups of Qingdai,there was no significant difference between the Qingdai high group and middle group(P>0.05).The IL-10 content of the Qingdai low group was significantly lower than the other two groups,and the difference was statistically significant(P<0.01)).Experimental results of the third paper:1.The effect of Qingdai on the relative expression of PI3K and AktmRNA in HCoEpiC cellular inflammation modelCompared with the control group,the relative expression of PI3K m RNA in the model group was significantly increased,and the difference was statistically significant(P<0.05).There was no significant difference in the relative expression of PI3K m RNA between the model group,SASP group and each group of Qingdai,and the difference was not statistically significant(P>0.05).There was no significant difference in the relative expression of PI3K m RNA between each dose of Qingdai,and the difference was not statistically significant(P>0.05).2.The influence of Qingdai on the expression of PI3K,Akt and p-Akt protein in HCoEpiC cellular inflammation modelCompared with the control group,the relative expression of PI3K protein in HCoEpiC cells in the model group was significantly increased,and the difference was statistically significant(P<0.01).Compared with the model group,the relative expression of PI3K protein in the SASP group,the Qingdai high group,and the Qingdai middle group were significantly reduced,and the difference was statistically significant(P<0.01).Compared with the SASP group,the relative expression of PI3K protein in the Qingdai middle group was significantly increased,and the difference was statistically significant(P<0.05),while the difference between the high group of Qingdai and the low group of Qingdai was not significant(P>0.05).Compared with the high group of Qingdai,the relative expression of PI3K protein in the middle group of Qingdai increased significantly,and the difference was statistically significant(P<0.05).The difference between the Qingdai middle group and the Qingdai low group was not obvious,and the difference was not statistically significant(P>0.05).The relative expression of AKT protein in HCoEpiC cells did not change significantly in each group.Compared with the control group,the relative expression of p-Akt/Akt protein in HCoEpiC cells of the model group was significantly increased,and the difference was statistically significant(P<0.01).Compared with the model group,the relative expression of p-Akt/Akt protein in HCoEpiC cells in the SASP group and the Qingdai group was significantly reduced,and the difference was statistically significant(P<0.01,P<0.05).Compared with the SASP group,there was no significant difference in the relative expression of p-Akt/Akt protein between the high,middle and low dose groups of Qingdai(P>0.05).Compared with the high Qingdai group,the relative expression of p-Akt/Akt protein in the low Qingdai group increased,and the difference was statistically significant(P<0.05).There was no significant difference in the relative expression of p-Akt/Akt protein between the middle group of Qingdai and the low group of Qingdai,and there was no statistical significance(P>0.05).Experimental results of the forth paper:1.The effect of Qingdai on the relative expression of IKKα,IκBα,and NF-κB p65m RNA in the HCoEpiC cellular inflammation modelFrom the results of IKKαand IκBα,it can be seen that drug intervention did not affect the two.The relative expression of IKKαand IκBαm RNA in each group did not differ significantly,and the difference was not statistically significant(P>0.05).Compared with the control group,the relative expression of NF-κB p65 m RNA in HCoEpiC cells in the model group was significantly increased,and the difference was statistically significant(P<0.01).After drug intervention,the relative expression of NF-κB p65 m RNA in the SASP group and each dose group of Qingdai decreased significantly,and the difference was statistically significant(P<0.05).However,compared with the SASP group,there was no significant difference in the relative expression of NF-κB p65 m RNA between the high,medium and low dose groups of Qingdai,and the difference was not statistically significant(P>0.05).There was no significant difference between the groups of Qingdai,and it was not statistically significant(P>0.05).2.The effect of Qingdai on the expression of NF-κB p65 protein in HCoEpiC cellular inflammation modelCompared with the control group,the relative expression of NF-κB p65 protein in HCoEpiC cells in the model group was significantly increased,and the difference was statistically significant(P<0.05).After drug intervention,compared with the model group,the relative expression of NF-κB p65 protein in the SASP group,the Qingdai high group,and the Qingdai low group decreased,and the difference was statistically significant(P<0.05).There was no significant difference in the changes between each dose group of Qingdai and the SASP group(P>0.05).3.The effect of Qingdai on the expression of IKKαand p-IKKαprotein in HCoEpiC cellular inflammation modelCompared with the control group,the p-IKKα/IKKαprotein expression of HCoEpiC cells in the model group was significantly increased,and the difference was statistically significant(P<0.01).After drug intervention,the expression of p-IKKα/IKKαprotein in the SASP group,the Qingdai high group and the middle group decreased significantly,and the difference was statistically significant(P<0.01,P<0.05).Compared with the SASP group,there was no significant difference in the expression of p-IKKα/IKKαprotein between the Qingdai high and middle groups(P>0.05),while the expression of p-IKKα/IKKαin the low Qingdai group was significantly increased,and the difference was statistically significant(P<0.05).There was no statistically significant difference in the expression of p-IKKα/IKKαprotein between the high and middle groups(P>0.05),but the expression was lower than that of the Qingdai low group,and the difference was statistically significant(P<0.05).4.The effect of Qingdai on the expression of IκBαand p-IκBαprotein in HCoEpiC cellular inflammation modelCompared with the control group,the p-IκBα/IκBαprotein expression of HCoEpiC cells in the model group was significantly increased,and the difference was statistically significant(P<0.01).After drug intervention,the expression of p-IKKα/IKKαprotein in the SASP group,Qingdai high group and middle group significantly decreased,and the difference was statistically significant(P<0.01).Compared with the SASP group,there was no significant difference in the expression of p-IκBα/IκBαprotein between the high and middle groups of Qingdai(P>0.05),while the expression of low group increased,and the difference was statistically significant(P<0.01).The expression of p-IκBα/IκBαprotein in the three dose groups of Qingdai was negatively correlated with the increase in the concentration of Qingdai,and the difference in p-IκBα/IκBαprotein expression between the two groups was statistically significant(P<0.01,P<0.05).Conclusion:1.LPS-induced HCoEpiC cells can cause cellular inflammation models.When the concentration of LPS is 10μg/mL and the culture time is 24h,the content of inflammatory factors in the cells is higher.2.Qingdai can significantly reduce the expression of pro-inflammatory cytokines IL-1β,IL-6,IL-12,and TNF-αand increase the expression of anti-inflammatory cytokines IL-4and IL-10.3.The anti-inflammatory effect of Qingdai may be related to the inhibition of key signaling molecules of PI3K and Akt.By inhibiting the expression of PI3K and Akt,the downstream inflammatory cytokines can be inhibited.4.In the process of regulating inflammatory cytokines,Qingdai is related to the inhibition of the related activities of IKK,IκB and NF-κB in NF-κB,a key pathway of inflammation regulation. |
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