| Objective:The level of pericentriolar material 1(PCM1)in the colonic mucosa of ulcerative colitis(UC)patients with different disease severity was detected,to explore the correlation between PCM1 and UC at the clinical level.At the cellular level,the mechanism of PCM 1 in inflammatory response and immune regulation was explored.Further,the correlations between the pathway factors involved in the above mechanisms and UC were verified in clinical samples.This study aimed to reveal the mechanism of PCM 1 in UC,and to find a novel therapeutic target for UC treatment.Methods:This study is divided into three parts:Part ?(clinical level):1.Colonic mucosa were collected from 30 healthy controls and 98 UC patients,including 32 mild UC patients,34 moderate UC patients,and 32 severe UC patients.The expression and localization of PCM1 in the colonic mucosa were detected by immunohistochemistry(IHC),to preliminarily explore the correlation between PCM1 and UC.2.Colonic mucosa were collected from 10 healthy controls,10 mild UC patients,10 moderate UC patients and 10 severe UC patients.The transcription and expression levels of PCM 1 in colonic mucosa were detected by qRT-PCR and Western blotting to further verify the correlation between PCM1 and UC.Part ?(cellular level):1.To investigate the role of PCM 1 in inflammatory response1.1 The inflammatory conditions of THP-1 cells and Caco-2 cells were constructed with lipopolysaccharide(LPS)under gradient concentrations,the level of PCM1 protein was detected by Western blotting to explore the correlation between PCM1 and inflammation.1.2 THP-1 cell lines and Caco-2 cell lines with stable expression or interference of PCM1 gene were constructed.1.3 The expression level of PCM1 was reduced and overexpressed in THP-1 cells and Caco-2 cells,and LPS was used to stimulate the cells to construct inflammatory conditions.The levels of proinflammatory cytokines(TNF-?,IL-1?,and IL-18)and anti-inflammatory cytokines(TGF-? and IL-4)in the supernatants were detected by ELISA to clarify the role of PCM 1 in the inflammatory response.2.To study the immune pathway of PCM 1 in regulating inflammation Based on the clues provided by the previous results,the mechanism of PCM 1 in immune regulation was studied,and relevant verification experiments were carried out.2.1 In THP-1 cells,PCM1 levels were interfered and overexpressed,then cells were treated with LPS and ATP.The effects of PCM 1 on NLRP3 inflammasomes(NLRP3,ASC and caspase-1)were detected by Western blotting.2.2 In THP-1 cells,PCM1 levels were interfered and overexpressed,LPS and ATP were used to construct pyroptosis model.The expression level of pyroptosis mediated protein(gasdermin D)was detected by Western blotting.The concentrations of pyroptosis related cytokines(IL-1? and IL-18)in supernatants were detected by ELISA.To clarify the effect of PCM1 on pyroptosis.2.3 In THP-1 cells,PCM1 levels were interfered and overexpressed,LPS and ATP were used to construct pyroptosis model.Three techniques were used to verify the effect of PCM1 on pyroptosis from different perspectives.(1)TUNEL staining detected the effect of PCM 1 on cell DNA damage.(2)PI staining detected the effect of PCM 1 on cell membrane integrity.(3)LDH release assay detected the effect of PCM 1 on cell membrane permeability.Part ?(clinical level):1.The ultrastructural changes and pyroptosis levels of colonic mucosa in healthy controls and UC patients with different disease severity were observed by transmission electron microscope(TEM).2.10 healthy controls,10 mild UC patients,10 moderate UC patients,and 10 severe UC patients were enrolled,and their colonic mucosa was collected to make paraffin blocks and sections.TUNEL staining was used to detect DNA damage in the colonic mucosa.IHC was used to detect the expression and localization of proteins in the PCM 1-activating NLRP3/caspase-1/gasdermin D/IL-1? and IL18 pathway in the colonic mucosa.3.10 healthy controls and 10 UC patients were enrolled,and their colonic mucosa was collected to extract total RNA and total proteins.The transcription and expression levels of factors in the PCM 1-activating NLRP3/caspase-1/gasdermin D/IL-1?and IL18 pathway were detected by qRT-PCR and Western blotting.Results:Part I(clinical level):1.IHC showed that PCM1 was expressed in the cytoplasm of epithelial cells and inflammatory cells of colonic mucosa,and strongly expressed in the germinal center of intestinal mucosal lymphoid follicles.The proportion of PCM1 positive cells in the healthy controls,mild UC patients,moderate UC patients,and severe UC patients were(0.12±0.17),(1.31±1.59),(0.97±1.54),and(0.27±0.38),respectively.There were significant differences in the expression levels of PCM1.F(P)=7.711(<0.0001).Compared with healthy controls,the levels of PCM1 in the colonic mucosa of UC patients were all significantly increased(P<0.05),and the level of PCM1 in the intestinal mucosa of severe UC patients was lower than that in the mild UC patients(P<0.01).2.The qRT-PCR and Western blotting tests showed that compared with healthy controls,the mRNA and protein levels of PCM 1 in the colonic mucosa of UC patients were increased.Further analysis of the correlation between PCM1 and the severity of UC showed that the level of PCM1 was highest in mild UC and moderate UC,and PCM1 showed a downward trend in severe UC.The above results were statistically significant(P<0.05).Part II(cellular level):1.In THP-1 cells and Caco-2 cells,the expression levels of PCM1 increased with the increase of LPS concentrations,and PCM1 decreased after reaching the expression peak.2.THP-1 cell lines and Caco-2 cell lines with stable expression or interference of PCM1 gene were successfully constructed.3.In the LPS-induced THP-1 and Caco-2 cells,PCM1 promoted the secretion of proinflammatory cytokines(TNF-?,IL-1?,and IL-18)and inhibited the secretion of anti-inflammatory cytokines(TGF-? and IL-4),and this trend was more significant in THP-1 cells,suggesting that PCM1 promoted inflammatory response.4.In LPS+ATP induced THP-1 cells,interference of PCM1 decreased expression levels of NLRP3,caspase-1,and cleaved caspase-1.Conversely,overexpression of PCM1 increased expression levels of NLRP3,caspase-1,and cleaved caspase-1.It suggested that PCM1 could promote the activation of NLRP3 inflammasomes.5.In THP-1 cells,the pyroptosis model was established by LPS+ATP treatment.Interference of PCM1 reduced the expression levels of gasdermin D and gasdermin D-N,and inhibited the release of IL-1? and IL-18.On the contrary,overexpression of PCM1 increased the expression levels of gasdermin D and gasdermin D-N,and promoted the release of IL-1? and IL-18.It is suggested that PCM1 may trigger gasdermin D-mediated pyroptosis.6.In THP-1 cells,the pyroptosis model was established by LPS+ATP treatment.TUNEL staining showed the proportion of TUNEL positive cells was increased after the addition of LPS+ATP(P<0.001).The proportion of TUNEL positive cells in the interfered PCM1 group(shPCM1+LPS+ATP)was lower than that in the interfered control group(shCtrl+LPS+ATP)(P<0.01).On the contrary,the proportion of TUNEL positive cells in the overexpressed PCM1 group(mPCM1+LPS+ATP)was higher than that in the overexpressed control group(mCtrl+LPS+ATP)(P<0.001).The proportion of TUNEL positive cells was decreased by adding the pyroptosis inhibitor NSA(mPCM1+LPS+ATP+NSA vs mPCM1+LPS+ATP,P<0.001).It was suggested that PCM1 could aggravate DNA damage and pyroptosis.7.PI staining showed the proportion of PI positive cells increased after adding LPS+ATP(P<0.05).The proportion of PI positive cells in the interfered PCM1 group(shPCM1+LPS+ATP)was lower than that in the interfered control group(shCtrl+LPS+ATP)(P<0.001).On the contrary,the proportion of PI positive cells in the overexpressed PCM1 group(mPCM1+LPS+ATP)was higher than that in the overexpressed control group(mCtrl+LPS+ATP)(P<0.05).The proportion of PI positive cells could be decreased by NSA(mPCMl+LPS+ATP+NSA vs mPCM1+LPS+ATP,P<0.001).It was suggested that PCM1 could destroy the integrity of cell membrane and aggravate pyroptosis.8.LDH release assay showed that LDH release was significantly increased after adding LPS+ATP(P<0.001).LDH release in the interfered PCM1 group(shPCM1+LPS+ATP)was lower than that in the interfered control group(shCtrl+LPS+ATP)(P<0.001).The release of LDH could be decreased by adding NSA(mPCM1+LPS+ATP+NSA vs mPCM1+LPS+ATP,P<0.001).It is suggested that reducing the level of PCM 1 can reduce the permeability of cell membrane and reduce pyroptosis.Part ?(clinical level):1.TEM showed that compared with healthy controls,morphology changes of epithelial cells and inflammatory cells in colonic mucosa of UC patients had occurred.With the aggravation of the UC severity,the damage of intestinal barrier structure was aggravated,and the level of pyrotosis was increased.2.TUNEL staining showed that compared with healthy controls,the proportion of TUNEL positive cells in colonic mucosa of UC patients was increased(P<0.05).The proportion of TUNEL positive cells in colonic mucosa of severe UC patients was higher than that of mild UC patients(P<0.05).It suggested that DNA damage and pyroptosis occurred in the colonic mucosa of UC patients,and the pyroptosis level was the highest in patients with severe UC.3.IHC showed that NLRP3,caspase-1,gasdermin D,IL-1?,and IL-18 were expressed in glandular epithelial cells and inflammatory cells of colonic mucosa.Among them,caspase-1,gasdermin D,IL-1?,and IL-18 were strongly expressed in crypt abscess,caspase-1 and IL-1? were strongly expressed in the germinal center of lymphoid follicle.According to the statistics of the proportion of IHC positive cells,the expression levels of NLRP3,caspase-1,gasdermin D,IL-1? and IL-18 in the colonic mucosa of UC patients were significantly increased compared with the healthy controls(all P<0.05).4.qRT-PCR showed that the transcriptional levels of NLRP3,caspase-1,gasdermin D,IL-1? and IL-18 in the colonic mucosa of UC patients were significantly increased compared with healthy controls(all P<0.05).5.Western blotting showed that protein levels of NLRP3,cleaved caspase-1,gasdermin D-N,IL-1? and IL-18 in colonic mucosa of UC patients were significantly increased compared with healthy controls(all P<0.05).Conclusions:1.The expression level of PCM 1 in colonic mucosa of UC patients was significantly increased.PCM1 was mainly expressed in epithelial cells and inflammatory cells of colonic mucosa,and strongly expressed in the germinal center of lymphatic follicles.Suggesting that PCM1 may be involved in the inflammatory response and immune regulation of UC.2.In THP-1 cells and Caco-2 cells,the expression levels of PCM1 were concentration-dependent with LPS.In the LPS-induced inflammation condition of THP-1 and Caco-2 cells,PCM1 promoted the secretion of proinflammatory cytokines and inhibited the secretion of anti-inflammatory cytokines,suggesting PCM 1 promoted the inflammatory response.In LPS+ATP-induced THP-1 pyroptosis model,PCM1 promoted pyroptosis by activating the NLRP3/caspase-1/gasdermin D/IL-1? and IL18 immune pathway.3.The colonic mucosa of UC patients occurred pyroptosis and DNA damage.The expression levels of factors in the PCM 1-activating NLRP3/caspase-1/gasdermin D/IL-1? and IL18 pathway were increased in the colonic mucosa of UC patients.Final conclusion:Aberrant expression of PCM1 activated NLRP3 inflammasome,triggered gasdermin D-mediated pyroptosis,increased the release of IL-1? and IL-18,and ultimately promoted the inflammation of UC. |
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