| Object:Explore whether Sishen pills(SSP)can regulate the inflammatory differentiation of dendritic cells through mi R-505-3p,thus treating chronic ulcerative colitis.Methods:40 SPF-grade Balb/c mice were randomly divided into 4 groups of 10 mice each,set up as normal group(Control),normal+SSP group(Ctrl+SSP),model group(DSS),and model+SSP group(DSS+SSP),respectively.Dextran sodium sulfate(DSS)was used to induce chronic ulcerative colitis model in mice in 3 stages,with mice in Control and Ctrl+SSP groups drinking distilled water freely throughout,and in DSS and DSS+SSP groups altogether,the first stage was given 3%DSS solution for 7days,the second stage was changed to distilled water for 7 days,and the third stage was changed to 2%DSS solution for 7 days.DSS solution for 7 days.On the first day of the second stage of model induction,the Ctrl+SSP and DSS+SSP groups were given 5 mg/m L of Sishen pills suspension by gavage according to the body weight of the mice,and the remaining two groups were given the same volume of distilled water by gavage once a day at a fixed time,and the mice were given the drug by gavage for14 days.During the replication process,FOB fecal occult blood test and DAI score were used to assess the disease status of the mice.If the FOB fecal occult blood test was positive or the DAI score was significantly different from that of the normal group for 3 consecutive days,the mouse ulcerative colitis model was considered to be successfully replicated and could be included in the sample for subsequent experimental analysis;otherwise,the sample was rejected.The mice were executed on the next day after the experiment,and the spleen and colon tissues were dissected and separated for testing.The mouse spleen weight,colon length and colon weight were observed,weighed and recorded,and the mouse colon index and colon weight per unit length were calculated;HE-stained pathological sections of colon tissues were prepared,observed and scored for pathological histology;interleukin 6(IL-6),interleukin 1β(IL-1β)and tumor necrosis factorα(TNF-α)were detected in colon tissues by enzyme-linked immunosorbent assay(ELISA);flow cytometry(FCM)to detect the expression levels of dendritic cells and their subpopulations in spleen;whole transcriptomics was used to sequence differential RNAs and genes in mouse colon tissues,and fluorescence quantitative PCR(RT-PCR)was used to detect the expression of key regulatory mi RNAs in colon tissues to validate the sequencing results.Mouse bone marrow-derived dendritic cells(BMDCs)were isolated,purified and induced,and their colony status were observed by light microscopy,also,the maturation status was detected by FCM.The IC50 and safe dose range of Sishen pills on BMDCs were determined using cell viability assay(Cell counting kit-8,CCK8).After collecting the identified and qualified immature dendritic cells,the normal group(Vehicle),the model group(LPS),the model+Sishen pills high dose group(LPS+SSP-H),the model+Sishen pills medium dose group(LPS+SSP-M),and the model+Sishen pills low dose group(LPS+SSP-L)were set up,and the high,medium,and low dose were set to 6 mg/m L,4 mg/m L,and 4 mg/m L respectively,according to the results of the pre-experiment.Firstly,these three doses groups were pre-administered for 20 h,subsequently,lipopolysaccharide(LPS,055:B5,100ng/m L)was added for 4 h to induce mature differentiation of dendritic cells toward the inflammatory state,then,the cell proliferation and apoptosis,as well as the expression of co-stimulatory molecules on the surface of dendritic cells were detected by FCM.Besides,the secretion level of inflammatory cytokines IL-6,IL-1βand TNF-αof BMDCs was measured by ELISA.Similarly,the immature dendritic cells that were successfully identified and qualified were collected.The relative expression of mi R-505-3p and gene CDH1 on BMDCs was detected by RT-PCR,and the protein expression of E-cadherin on BMDCs was detected by Western blotting(WB).BMDCs with knockdown expression of mi R-505-3p were constructed by lentivirus transfection,and the knockdown effect of mi R-505-3p was verified by RT-PCR and WB;After obtaining BMDCs with low expression of mi R-505-3p,set the normal group(Vehicle),model group(LPS),model+Sishen pills group(LPS+SSP).According to the previous experimental results,the drug dose of 4 mg/m L Sishen pills was selected as the experimental dose,FCM was used to detect cytokines related to inflammatory differentiation of BMDCs with low expression of mi R-505-3p,and ELISA was used to detect the secretion levels of inflammatory cytokines IL-6,IL-1βand TNF-αof BMDCs with low expression of mi R-505-3p.Results:1.Liquid chromatography-mass spectrometry analysis of Sishen pillsAfter combining the results of three chromatographic screenings and comparing with the database,more than 4000 compounds were detected,including mainly atovaquone,glycyrrhiza spp.A,leucine,myristic acid,wogonin and eugenol,etc.According to literature research,the above active substances have the effects of anti-microbial infection,anti-cancer,anti-inflammation and inhibition of key proteins of pathways related to inflammatory bowel disease.2.Sishen pills effectively alleviates DSS-induced chronic ulcerative colitis in miceIn the Control group and Ctrl+SSP group,the mice gained weight steadily and were in good condition;in the DSS group,the mice lost weight,had poor vitality,had disorganized fur,had loose stools or even blood in stool,and were found to have shortened colonic length,increased colonic edema and weight,and microscopically,the inflammatory infiltration in the lamina propria of the colon was obvious,the crypt disappeared,and the ulcer was obvious.After continuous administration of Sishen pills,in DSS+SSP group,the body weight lost due to modeling recovered gradually,the length of colon returned to close to the normal level,and the weight of colon and intestinal weight index decreased significantly,and the microscopic inflammatory infiltration of colon was reduced,ulcers were reduced,and the pathological histological score decreased significantly(P<0.01).expression levels were significantly increased in the DSS group(P<0.01),suggesting the occurrence of colonic inflammation,while the expression levels of the above inflammatory factors were significantly decreased in the DSS+SSP group(P<0.05 or P<0.01),suggesting that Sishen pills could effectively alleviate the inflammation of chronic ulcerative colitis in mice.3.Sishen pills significantly regulates the inflammatory differentiation status of mouse dendritic cells and their subpopulations.The results of flow cytometry suggest that compared with the Control group,the inflammatory dendritic cell populations in the DSS group,which included:CD11c~+CD103~+E-cadherin~+DC,CD11c~+CD103~+CD172α~+DC,CD11c~+CD103~+F4/80~+DC,CD11c~+CD103~+Ly6c~+DC,CD11c~+CD103~+MHC-Ⅱ~+DC,CD11c~+CD103~+CD64~+DC and CD11c~+CD103~+FcεRI-α~+DC,increased significantly(P<0.01).The expression levels of other cell surface markers related to dendritic cell homeostasis,proliferation,and development,including CD115,Flt3,and CD8α,were significantly increased(P<0.01),while CD24,which inhibits dendritic cell activation,was significantly decreased in the DSS group(P<0.01).After Sishen pills administration,compared with DSS group,the expression levels of CD11c~+CD103~+E-cadherin~+DC,CD11c~+CD103~+CD172α~+DC,CD11c~+CD103~+F4/80~+DC,CD11c~+CD103~+Ly6c~+DC,CD11c~+CD103~+MHC-Ⅱ~+DC,CD11c~+CD103~+CD64~+DC,CD11c~+CD103~+FcεRI-α~+DC in DSS+SSP group were decreased significantly(P<0.01),the expression levels of CD8α,CD115 and Flt3 were also decreased significantly(P<0.01),while the expression level of CD24 was significantly restored(P<0.05).These results indicate that Sishen pills can effectively inhibit the activation and maturation of dendritic cells and inflammatory dendritic cells to relieve UC in mice.4.Whole-transcriptome sequencing demonstrates that Sishen pills regulates key RNAs and genes in mouse colon tissueAccording to the significance level p adjust<0.01 and 4.0-fold difference in up/down regulation,298 differential m RNAs(201 up-regulated,97 down-regulated),42 differential mi RNAs(18 up-regulated,24 down-regulated),234 differential lnc RNAs(184 up-regulated,50 down-regulated),10 differential circ RNAs(9up-regulated,1 down-regulated).In the differential analysis of DSS group vs DSS+SSP group,18 differential m RNAs(10 up-regulated,8 down-regulated),15differential mi RNAs(8 up-regulated,7 down-regulated),88 differential lnc RNAs(27up-regulated,61 down-regulated),and 3 differential circ RNAs(1 up-regulated,2down-regulated).The RNAs that were effectively regulated(i.e.,the treatment group could reverse the model group)after Sishen pills intervention were screened by Venn diagram.Based on the above effectively regulated RNAs,the ce RNA network was constructed and the core key mi RNAs were screened:mi R-380-5p,mi R-490-3p,X-38964,mi R-335-3p,mi R-10b-3p,mi R-485-3p,mi R-485-5p,mi R-370-3p,mi R-9-3p,mi R-504-5p,mi R-505-3p,etc.Among them,it is known from literature research that mi R-505-3p shares regulatory transcription factors with E-cadherin,a key protein of inflammatory dendritic cells,and mi R-505-3p may regulate the inflammatory differentiation state of dendritic cells through regulation.5.Sishen pills effectively inhibits LPS induced inflammatory responses in mouse bone marrow-derived dendritic cells(BMDCs)BMDCs were extracted,separated,induced and purified from mouse bone marrow.The immature BMDCs colonies were obviously observed by optical microscopy,and the dendritic morphology of mature BMDCs was significant.Flow cytometry showed that the expression level of surface costimulatory molecules of BMDCs increased significantly after maturation.CCK8 cytotoxicity test showed that the IC50 of Sishen pills on BMDCs was 37.10 mg/m L at 24 h,19.21 mg/m L at 48 h,and 19.17 mg/m L at 72 h.When the dose of Sishen pills exceeded 8 mg/m L,the cell viability of BMDCs decreased significantly.It showed that Sishen pills began to show toxicity to BMDCs,so the dose of subsequent experiments was selected below 8mg/m L.Flow cytometry showed that Sishen pills could accelerate the apoptosis of BMDCs and slow down the proliferation of BMDCs.By detecting surface costimulatory molecules on BMDCs,Sishen pills can reduce the maturation and inflammatory differentiation of BMDCs.ELISA results showed that Sishen pills could effectively inhibit the secretion of inflammatory cytokines IL-6,IL-1βand TNF-αby BMDCs.6.Sishen pills inhibit inflammatory differentiation of dendritic cells by regulating mir-505-3pRT-PCR showed that the relative expression level of mi R-505-3p was significantly increased after LPS-induced BMDCs,and significantly decreased after Sishen pills intervention.The relative expression of CDH1(E-cadherin protein encoding gene)on BMDCs increased significantly in the model group,and the trend was reversed by the intervention of Sishen pills.At the same time,the expression level of E-cadherin protein on BMDCs was verified by WB,which was similar to the trend of its gene expression.The expression level of E-cadherin protein was significantly increased in the model group,but significantly decreased in the Sishen pills group.MOI=100 and transfection time 72 h were selected as the lentivirus transfection conditions to construct BMDCs with low expression of mi R-505-3p.Subsequently,RT-PCR was used to verify that the significant reduction of mi R-505-3p in BMDCs after transfection knockdown indicated successful transfection.The protein expression of E-cadherin in transfected BMDCs was significantly decreased by WB detection.The above results suggest that the expression trend of mi R-505-3p and E-cadherin in BMDCs is consistent.Subsequent flow cytometry showed that after knockdown of mi R-505-3p,CD86,MHC-Ⅱ,TNF-α,IL-12 and other inflammatory response related indexes of BMDCs in LPS group were still significantly increased,and IL-10 levels were significantly decreased,but Sishen pills could not reverse the trend of LPS group.Similarly,ELISA detected the levels of inflammatory cytokines IL-6,IL-1β,and TNF-αsecreted by BMDCs after knockdown of mi R-505-3p.IL-6,IL-1β,and TNF-αwere still significantly increased in LPS group,but Sishen pills could not reverse the trend of LPS group.These results indicate that one of the possible mechanisms of Sishen pills in the effective treatment of ulcerative colitis in mice is to inhibit the expression of E-cadherin protein on dendritic cells by regulating mi R-505-3p,so as to affect the differentiation of dendritic cells to inflammatory state.Conclusions:Sishen pills can alleviate symptoms of chronic ulcerative colitis in mice by affecting the inflammatory differentiation of dendritic cells,and the mechanism is mainly through regulating mi R-505-3p to inhibit E-cadherin protein expression on dendritic cells,which can affect the differentiation of dendritic cells into inflammatory state. |
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