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Department of Critical Care Medicine, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu, ChinaAnhui Province Clinical Research Center for Critical Care Medicine (Respiratory Disease), Wuhu, China
Department of Critical Care Medicine, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu, ChinaAnhui Province Clinical Research Center for Critical Care Medicine (Respiratory Disease), Wuhu, China
Department of Critical Care Medicine, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu, ChinaAnhui Province Clinical Research Center for Critical Care Medicine (Respiratory Disease), Wuhu, China
Department of Critical Care Medicine, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu, ChinaAnhui Province Clinical Research Center for Critical Care Medicine (Respiratory Disease), Wuhu, China
Address correspondence to Wei-Hua Lu, M.D., Department of Critical Care Medicine, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), No. 2 Zheshan Rd., Wuhu 241001, Anhui, China.
Department of Critical Care Medicine, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu, ChinaAnhui Province Clinical Research Center for Critical Care Medicine (Respiratory Disease), Wuhu, China
The intestines play a crucial role in the development of sepsis. The balance between autophagy and apoptosis in intestinal epithelial cells is dynamic and determines intestinal permeability. The present study focused on the potential role of autophagy in sepsis-induced intestinal barrier dysfunction and explored the mechanisms in vivo and in vitro. Excessive apoptosis in intestinal epithelia and a disrupted intestinal barrier were observed in septic mice. Promoting autophagy with rapamycin reduced intestinal epithelial apoptosis and restored intestinal barrier function, presenting as decreased serum diamine oxidase (DAO) and fluorescein isothiocyanate–dextran 40 (FD40) levels and increased expression of zonula occludens-1 (ZO-1) and Occludin. Polo-like kinase 1 (PLK1) knockdown in mice ameliorated intestinal epithelial apoptosis and the intestinal barrier during sepsis, whereas these effects were reduced with chloroquine and enhanced with rapamycin. PLK1 also promoted cell autophagy and improved lipopolysaccharide-induced apoptosis and high permeability in vitro. Moreover, PLK1 physically interacted with mammalian target of rapamycin (mTOR) and participated in reciprocal regulatory crosstalk in intestinal epithelial cells during sepsis. This study provides novel insight into the role of autophagy in sepsis-induced intestinal barrier dysfunction and indicates that the PLK1-mTOR axis may be a promising therapeutic target for sepsis.
Sepsis, one of the most acute and serious disease complications in the intensive care unit, is usually caused by various infections and results in life-threatening organ dysfunction.
Despite the increased understanding of sepsis pathophysiology and the application of advanced clinical treatments, sepsis remains a major cause of health loss worldwide, with a high health-related burden.
However, the underlying mechanisms remain rather vague and are worthy of further exploration.
Autophagy, a highly conserved biological phenomenon in all eukaryotic cells, is defined as a catabolic process that transfers damaged organelles, invading pathogens, or aggregated proteins to the lysosome for degradation.
Autophagy within the epithelium is involved in regulating the balance between the gut microbiota and resident immune cells, and in the host defense against intestinal infections.
Mammalian target of rapamycin (mTOR), an upstream negative regulator of autophagy, is a highly conserved serine/threonine protein kinase that regulates organism growth and homeostasis.
mTOR is present in two multiprotein complexes termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which are structurally and functionally distinct.
Accumulating evidence implies that activation of mTORC1 accompanied by activation of its downstream target ribosomal protein S6 (RPS6) is associated with activation of massive and complex signaling pathways, as well as inflammatory responses.
However, the functions of mTOR and autophagy, and the mechanisms by which they interact in the pathogenesis of sepsis-induced intestinal barrier dysfunction, especially in intestinal epithelial cells, remain unclear and are worthy of investigation.
Polo-like kinase 1 (PLK1) is another serine/threonine protein kinase that drives cell proliferation by promoting mitosis and cytokinesis.
PLK1 protects against sepsis-induced intestinal barrier dysfunction by regulating the balance between proliferation and apoptosis in the intestinal epithelium,
although little is known about the link between PLK1 and autophagy in the intestinal epithelium.
In the current study, mice were subjected to cecal ligation and puncture (CLP) to establish a sepsis model in vivo, and autophagy was subsequently evaluated. The effects of rapamycin on intestinal barrier function were also investigated. Additionally, the relationship between PLK1 and mTOR was explored. Results from this study clarify the role of autophagy in sepsis-induced intestinal barrier dysfunction and highlight the importance of the PLK1-mTOR axis in regulating autophagy during sepsis.
Materials and Methods
Animal Model
Healthy wild-type C57BL/6J mice (aged 8 to 10 weeks, weighing 25 ± 5 g, male) were obtained from Keygen Biotech (Nanjing, China). Rosa26-CAG-CreERT2;H11-Loxp-Stop-Loxp-PLK1 mice (PLK1 conditional knock-in mice, hereafter referred to as CAG-PLK1 mice) and CAG-CreERT2 mice were generated by Shanghai Model Organisms Center, Inc. (Shanghai, China). All mice were maintained on a 12-hour light/dark cycle in a specific pathogen-free animal room with access to water and a standard rodent diet. Tamoxifen (Sigma-Aldrich, St. Louis, MO) was dissolved in corn oil at a concentration of 20 mg/mL, and an intraperitoneal injection of 100 μL was administered once per day for a total of 5 consecutive days to induce conditional knock-in of PLK1 in mice. The experiments were started 1 week after the final injection. All experimental protocols were performed according to the Guide for the Care and Use of Laboratory Animals
Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council: Guide for the Care and Use of Laboratory Animals. Eighth Edition. National Academies Press,
Washington, DC2011
In brief, mice were anesthetized with 1% pentobarbital, a 0.5- to 1-cm midline abdominal incision was made, and the cecum was then exposed. Subsequently, the mesentery of the cecum was carefully separated, the cecum was ligated below the ileocecal valve in the distal three-quarters, and a single through-and-through perforation was made with a 21-gauge needle. After puncture, the cecum was returned to the abdomen, and the incision was sutured with sterile 3-0 silk. The sham-operated mice were subjected to laparotomy without CLP. Saline solution was injected subcutaneously (50 mg/kg) for resuscitation after surgery. In the treatment groups, rapamycin at 10 mg/kg bodyweight (s1039; Selleck, Houston, TX) or chloroquine at 60 mg/kg bodyweight (C6628; Sigma-Aldrich) was administered intraperitoneally 1 hour after the CLP operation based on previous studies.
At 24 hours after surgery, the mice were anesthetized, and blood was drawn by cardiac puncture. The mice were then euthanized, and the blood was centrifuged at 3000 × g for 15 minutes at 4°C. The supernatant was then collected and analyzed to assess the concentrations of diamine oxidase (DAO) using kits (A088-1-1; Jiancheng, Nanjing, China) according to the manufacturer's instructions.
Fluorescein Isothiocyanate–Dextran Assay in Vivo
The intestinal permeability in vivo was evaluated by the fluorescein isothiocyanate–dextran assay, which was performed as previously described.
In brief, a dose of 40 mg/100 g fluorescein isothiocyanate–dextran 40 (FD40; MKBio, Shanghai, China) was gavaged into mice 4 hours prior to the time that permeability was measured. The mice were then sacrificed, and the blood was collected and subsequently spun down at 5031 × g for 5 minutes at 4°C to isolate the serum. Finally, a fluorescence microplate reader was used with an excitation wavelength of 488 nm and emission wavelength of 525 nm to measure the serum absorbance value of FD40.
Hematoxylin and Eosin Staining
Small intestine tissues were collected 24 hours after CLP and fixed with 10% paraformaldehyde overnight. The samples were then embedded in paraffin and sliced into 5-μm-thick sections. For pathological examination, the sections were stained with hematoxylin and eosin as previously described.
: grade 0—normal mucosal villi; grade 1—development of subepithelial Gruenhagen's space, usually at the apex of the villus; often with capillary congestion; grade 2—extension of the subepithelial space with moderate lifting of epithelial layer from the lamina propria; grade 3—massive epithelial lifting down the sides of villi. A few tips may be denuded; grade 4—denuded villi with lamina propria and dilated capillaries exposed. Increased cellularity of lamina propria may be noted; and grade 5—digestion and disintegration of lamina propria; hemorrhage and ulceration.
Histological Examination
For immunofluorescence staining, sections were blocked with fetal bovine serum for 30 minutes, washed with phosphate-buffered saline (PBS), and incubated sequentially with the primary antibody overnight at 4°C and with the secondary antibody at room temperature in the dark. The sections were then washed with PBS three times and imaged using a fluorescence microscope.
TUNEL Assay
For cell apoptosis analysis in the intestinal epithelium, a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed as described in the authors’ previous study.
In brief, tissue sections were incubated in permeabilization solution, stained with the reaction mixture, and subsequently incubated with DAPI. The number of TUNEL-positive cells was determined by counting the double-labeled cells in 10 areas of the section under a fluorescence microscope.
RNA Extraction and Real-Time PCR Analysis
Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, Carlsbad, CA), and cDNA was then created using a reverse transcription kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. Real-time PCR was performed on an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems, Waltham, MA) using a TaqMan PCR kit and SYBR Green dye (Invitrogen). The 2−ΔΔCt cycle threshold formula was used to calculate the relative abundance of transcripts. The primers used in this experiment are shown in Table 1.
Table 1Specific Primers Used to Amplify Target Genes in Real-Time PCR
The NCM460 cell line, a normal human colon mucosal epithelial cell line, was obtained from the Basic Medical College of Peking Union Medical College, Beijing. The human intestinal Caco-2 cell line was purchased from Cellcook Biotech (Guangzhou, China). Cells were cultured in RPMI 1640 medium or DMEM supplemented with 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2. When the cells were 70% to 80% confluent, they were exposed to various treatments at the indicated concentrations and for the indicated times. Vehicle-treated cells were used as control cells.
Plasmid and siRNA Transfection
The pCDNA3.1-PLK1 overexpression plasmid and PLK1 siRNA were constructed by Keygen Biotech, along with the corresponding controls. NCM460 cells were cultured in 6-well plates to 60% to 70% confluence, and serum-free Opti-MEM (Thermo Fisher Scientific) was then added to replace the original medium. Subsequently, the plasmid or siRNA was transfected into cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocols.
Measurement of TEER
Transepithelial electrical resistance (TEER) was measured to evaluate the effect of lipopolysaccharide (LPS) on cell permeability in vitro as previously described.
Briefly, Caco-2 cells were seeded on the upper layer of a Transwell 6-well plate (0.4 μm; Corning, Corning, NY), and different treatments were added according to the experimental design. An epithelial volt–ohm meter with a chopstick electrode (World Precision Inc., Sarasota, FL) was then used to measure the TEER every 1 hour.
Immunoblot Analysis
The small intestinal epithelium was isolated as previously described,
and total protein was extracted with a protein extraction kit (AMRESCO, Solon, OH). For extraction of total protein from cells, harvested cells were washed with cold PBS and lysed with lysis buffer containing protease and phosphatase inhibitor cocktails. The protein concentration was quantified with a BCA protein assay kit (Keygen Biotech). Subsequently, equal amounts of protein were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) at 100 V for 1 hour at 4°C. Membranes were blocked with 5% milk in Tris-buffered saline–Tween 20 and were then incubated with the indicated primary antibodies overnight. After washing with PBS three times, the membranes were incubated with the corresponding secondary antibodies. Finally, the membranes were analyzed using Super ECL Detection Reagent (Applygen, Beijing, China).
The following primary antibodies were used at the indicated dilutions: anti-PLK1 (1:1000; Abcam, Waltham, MA), anti–p-mTOR (1:500; Cell Signaling Technology, Danvers, MA), anti-mTOR (1:500; Cell Signaling Technology), anti–p-S6 (1:500; Cell Signaling Technology), anti-S6 (1:500; Cell Signaling Technology), anti-cleaved Caspase-3 (1:1000; Abcam), anti–Bcl-2 (1:1000; Abcam), anti-Bax (1:1000; Abcam), anti-LC3 (1:1000; Abcam), anti-Beclin1 (1:1000; Abcam), anti-P62 (1:1000; Abcam), anti–zonula occludens-1 (ZO-1) (1:500; Proteintech, Rosemont, IL), anti-Occludin (1:500; Proteintech), and anti-GAPDH (1:5000; Sigma-Aldrich).
Immunoprecipitation
To detect the endogenous interaction between PLK1 and mTOR, NCM460 cells were washed with ice-cold PBS and lysed in immunoprecipitation lysis buffer supplemented with phenylmethylsulfonyl fluoride (Sigma-Aldrich). The lysates were then centrifuged prior to incubation with Protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). The beads were then cleared by centrifugation. Lysates and immunoprecipitates were incubated with the indicated primary antibodies and the appropriate secondary antibody, and the complexes in the supernatants were separated and detected by SDS-PAGE.
Statistical Analysis
GraphPad Prism software version 5.0 (GraphPad Software Inc., San Diego, CA) was used for data analysis. Quantitative data are presented as the means ± SDs, and data were statistically evaluated using t-test or one-way analysis of variance. A value of P < 0.05 was considered statistically significant.
Results
Sepsis Induces Intestinal Barrier Dysfunction and Promotes Intestinal Epithelial Apoptosis in CLP Mice
To confirm the effect of sepsis on intestinal barrier function, the intestinal tissues of CLP mice were stained with hematoxylin and eosin. As visible in the sections, the intestinal villi in CLP mice were atrophic with a disordered structure (Figure 1A). The Chiu score, which indicates the severity of intestinal mucosal injury, was higher in CLP mice (Figure 1B).
Figure 1Sepsis induces intestinal barrier dysfunction and promotes intestinal epithelial apoptosis in cecal ligation and puncture (CLP) mice. Mice were sacrificed 24 hours after CLP and were then analyzed. A: Representative images of hematoxylin and eosin–stained sections from each group. The lower panels are enlarged images of the boxed areas in the upper panels. B: Chiu's score of hematoxylin and eosin–stained sections. C: The serum concentration of DAO in each group. D: Serum levels of FD40 in each group. E: The levels of zonula occludens-1 (ZO-1) and Occludin in the intestine were analyzed by Western blot analysis. F: Representative images of ZO-1 and Occludin staining in the intestine in each group are shown. G: Representative images of the TUNEL assay. The lower panels are enlarged images of the boxed areas in the upper panels. The graph shows the percentage of TUNEL-positive cells. H: The levels of apoptosis markers in the intestine were analyzed by Western blot analysis. The graph shows the relative band densities. Data are expressed as means ± SD. n = 3 independent experiments. ∗∗P < 0.01, ∗∗∗P < 0.001 versus the control group. Scale bars: 100 μm (A); 20 μm (A and G, lower); 50 μm (F and G, upper).
The levels of DAO and FD40 in serum, which indicate intestinal mucosal permeability, were increased in septic mice (Figure 1, C and D). However, the expression levels of the tight junction proteins ZO-1 and Occludin were decreased in CLP mice (Figure 1, E and F). The above results showed the disruption of the intestinal barrier during sepsis. Because excessive apoptosis in the intestinal epithelium contributes to the destruction of intestinal barrier integrity, intestinal epithelial apoptosis was assessed with a TUNEL assay, which showed that the number of TUNEL-positive cells was significantly increased in CLP mice (Figure 1G). Furthermore, proteins related to the apoptotic pathway were detected. In the intestinal epithelium of the CLP group, the levels of the proapoptotic proteins cleaved Caspase-3 and Bax were increased, whereas the level of the antiapoptotic protein Bcl-2 was decreased (Figure 1H). These results indicate excessive intestinal epithelial apoptosis in septic mice.
Rapamycin Promotes Autophagy and Reduces Apoptosis in Intestinal Epithelia in CLP Mice
Autophagy is a highly conserved and self-protective mechanism that coordinates multiple aspects of the cellular response to pathogens.
Thus, autophagic activity in the intestinal epithelium during sepsis was also evaluated in this study. Autophagosome formation, the initial step of autophagy, can be assessed by the conversion of cytosolic LC3-I protein to LC3-II protein. In this study, the level of LC3-II (Supplemental Figure S1) and the levels of Beclin1 and p62 (Supplemental Figure S1), two central regulators of autophagy, were not significantly changed in the CLP groups. Rapamycin, a select inhibitor of the mTOR pathway, was used to promote autophagy in mice. Under rapamycin treatment, the LC3-II/I ratio and Beclin-1 level were increased, and the expression of p62 was decreased in intestinal epithelial cells (Figure 2, A and B ), suggesting the activation of autophagy. Autophagy has been reported to be essential for protection against apoptosis in intestinal epithelial cells.
Therefore, intestinal epithelial apoptosis was assessed in CLP mice under rapamycin treatment. The number of TUNEL-positive cells was significantly increased in the CLP group but sharply decreased under rapamycin treatment (Figure 2C). Levels of cleaved Caspase-3 and Bax proteins were decreased, accompanied by an increased level of Bcl-2 protein, in the CLP + rapamycin mouse group compared with the CLP group (Figure 2D). The above results suggest that the proportion of apoptotic cells in the intestinal epithelium decreased after rapamycin application during sepsis.
Figure 2Rapamycin (Rapa) promotes autophagy and reduces apoptosis in intestinal epithelia in cecal ligation and puncture (CLP) mice. The mice in the rapamycin group were intraperitoneally injected with rapamycin (10 mg/kg bodyweight) 1 hour after the CLP operation; the mice were then sacrificed 24 hours after CLP. A: Representative images of LC3-II and P62 immunohistochemical staining in the intestine in each group are shown. B: The levels of autophagy markers in the intestine were analyzed by Western blot analysis. The graph shows the relative band densities. C: Representative images of the TUNEL assay in each group are shown. The lower panels are enlarged images of the boxed areas in the upper panels. The graph shows the percentage of TUNEL-positive cells. D: The levels of apoptosis markers in the intestine in each group were measured. The graph shows the relative band densities. Data are expressed as means ± SD. n = 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bars: 100 μm (A); 20 μm (C, lower); 50 μm (C, upper).
Rapamycin Alleviates Sepsis-Induced Intestinal Barrier Dysfunction in CLP Mice
The effect of rapamycin on intestinal barrier function was evaluated. Hematoxylin and eosin staining showed alleviation of intestinal villus abnormalities and a decreased Chiu's score in the CLP + rapamycin group compared with the CLP group (Figure 3, A and B ). In line with this observation, the elevated levels of serum DAO and FD40 in the CLP group were decreased following rapamycin injection (Figure 3, C and D). The decreased expression levels of the tight junction proteins ZO-1 and Occludin in the CLP group were also increased in the CLP + rapamycin group (Figure 3, E and F).
Figure 3Rapamycin (Rapa) alleviates sepsis-induced intestinal barrier dysfunction. The mice in the rapamycin group were intraperitoneally injected with rapamycin (10 mg/kg bodyweight) 1 hour after the cecal ligation and puncture (CLP) operation; the mice were then sacrificed 24 hours after CLP. A: Representative images of hematoxylin and eosin–stained sections in each group. The lower panels are enlarged images of the boxed areas in the upper panels. B: Chiu's score of hematoxylin and eosin–stained sections. C: The serum concentration of DAO in each group. D: Serum levels of FD40 in each group. E: The levels of ZO-1 and Occludin in the intestine were analyzed by Western blot analysis. The graph shows the relative band densities. F: Representative images of ZO-1 and Occludin staining in the intestine in each group are shown. Data are expressed as means ± SD. n = 3 independent experiments. ∗P < 0.05, ∗∗∗P < 0.001. Scale bars: 100 μm (A, upper); 20 μm (A, lower); 50 μm (F).
To determine whether the protective role of PLK1 relies on autophagy, CAG-PLK1 mice underwent CLP and were treated with chloroquine, an inhibitor of autophagy, or rapamycin. In the CAG-PLK1 mice, the LC3-II and Beclin1 levels were increased, whereas the expression of p62 was decreased, with elevated mRNA levels of LAMP2, which all indicated the activation of autophagy (Figure 4). Apoptosis in the intestinal epithelium was alleviated in septic CAG-PLK1 mice, with reduced TUNEL-positive cells, decreased levels of cleaved Caspase-3 and Bax, and increased expression of Bcl-2 (Figure 5). Furthermore, intestinal barrier function was improved in septic CAG-PLK1 mice, with a lower Chiu's score accompanied by decreased serum DAO and FD40 levels (Figure 6, A–C). The expression levels of the tight junction proteins ZO-1 and Occludin were also recovered in the septic CAG-PLK1 mice (Figure 6, D and E). However, these ameliorative phenomena in the CAG-PLK1 mice were deteriorated following chloroquine treatment, which demonstrated excessive apoptosis in the intestinal epithelia (Figure 5) and a disrupted intestinal barrier (Figure 6). By contrast, CAG-PLK1 mice treated with rapamycin presented activated autophagy (Figure 4), modified apoptosis of intestinal epithelia (Figure 5) and ameliorated intestinal barrier function (Figure 6). Taken together, these results indicate that PLK1 protects against sepsis-induced intestinal barrier dysfunction by promoting intestinal epithelial autophagy.
Figure 4PLK1 promotes intestinal epithelial autophagy in intestinal epithelia in cecal ligation and puncture (CLP) mice. CAG-PLK1 mice were intraperitoneally injected with chloroquine (CQ; 60 mg/kg bodyweight) or rapamycin (Rapa; 10 mg/kg bodyweight) 1 hour after the CLP operation; the mice were then sacrificed 24 hours after CLP. A: Representative images of LC3-II and P62 immunohistochemical staining in the intestine in each group are shown. B: The levels of autophagy markers in the intestine were analyzed by Western blot analysis. The graph shows the relative band densities. C: The mRNA expression of LAMP2 in each group is shown. Data are expressed as means ± SD. n = 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bars = 100 μm. WT, wild-type.
Figure 5PLK1 reduces apoptosis in intestinal epithelia in cecal ligation and puncture (CLP) mice. CAG-PLK1 mice were intraperitoneally injected with chloroquine (CQ; 60 mg/kg bodyweight) or rapamycin (Rapa; 10 mg/kg bodyweight) 1 hour after the CLP operation; the mice then were sacrificed 24 hours after CLP. A: Representative images of the TUNEL assay in each group are shown. The lower panels are enlarged images of the boxed areas in the upper panels. The graph shows the percentage of TUNEL-positive cells. B: The levels of apoptosis markers in the intestine in each group were measured. The graph shows the relative band densities. Data are expressed as means ± SD. n = 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bars: 50 μm (A, upper); 20 μm (A, lower). WT, wild-type.
Figure 6PLK1 protects against sepsis-induced intestinal barrier dysfunction by promoting intestinal epithelial autophagy. CAG-PLK1 mice were intraperitoneally injected with chloroquine (CQ; 60 mg/kg bodyweight) or rapamycin (Rapa; 10 mg/kg bodyweight) 1 hour after the cecal ligation and puncture (CLP) operation; the mice were then sacrificed 24 hours after CLP. A: Representative images of hematoxylin and eosin–stained sections in each group. The lower panels are enlarged images of the boxed areas in the upper panels. The Chiu's score of hematoxylin and eosin–stained sections is shown on the right. B: The serum concentration of DAO in each group. C: Serum levels of FD40 in each group. D: The levels of ZO-1 and Occludin in the intestine were analyzed by Western blot analysis. The graph shows the relative band densities. E: The mRNA expression of ZO-1 and Occludin in each group is shown. Data are expressed as means ± SD. n = 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scale bars: 100 μm (A, upper); 20 μm (A, lower). WT, wild-type.
PLK1 Promotes Cell Autophagy and Improves LPS-Induced Apoptosis and High Permeability in Vitro
To further verify the protective role of PLK1, Caco-2 cells were transfected with pcDNA-PLK1 and then incubated with LPS for 24 hours. The results showed that LPS treatment decreased the levels of LC3 II/I and Beclin1, and increased the expression of p62 (Figure 7A), indicating impaired autophagy. With pcDNA-PLK1 transfection, the expression levels of LC3 II/I, Beclin1, and p62 were all partly rescued. Moreover, overexpression of PLK1 also reduced LPS-induced cell apoptosis, as shown by the recovered expression of cleaved Caspase-3, Bax, and Bcl-2 (Figure 7B). The increased cell permeability, which was shown as increased expression of tight junction proteins (ZO-1 and Occludin) and decreased TEER levels with LPS treatment, was also reformative with pcDNA-PLK1 transfection (Figure 7C). With Si-PLK1 transfection, cell autophagy, cell apoptosis, and cell permeability were all deteriorative (Supplemental Figure S2). The above results indicate that PLK1 promotes cell autophagy and improves LPS-induced apoptosis and high permeability in vitro.
Figure 7PLK1 promotes cell autophagy and improves lipopolysaccharide (LPS)-induced apoptosis and high permeability in vitro. Caco-2 cells were transfected with pCDNA3.1-PLK1 (a PLK1 knock-in plasmid) or control pCDNA3.1 plasmid (empty vector) and then exposed to LPS (30 μg/mL) for 24 hours. A: The levels of autophagy markers were analyzed by Western blot. The graph shows the relative band densities. B: The levels of apoptosis markers in each group were measured. The graph shows the relative band densities. C: The levels of ZO-1 and Occludin were analyzed by Western blot analysis. The graph shows the relative band densities. D: Transepithelial electrical resistance (TEER) levels in each group. Data are expressed as means ± SD. n = 3 independent experiments. ∗∗P < 0.01, ∗∗∗P < 0.001.
Reciprocal Regulation of PLK1 and mTOR in Intestinal Epithelial Cells
Because the mTOR pathway plays a critical role in autophagy, to further explore whether PLK1 promotes autophagy via the mTOR pathway in intestinal epithelial cells, immunoprecipitation was performed to identify the physical interaction between PLK1 and mTOR in vitro. Normal human colonic epithelial cells, NCM460, were selected as an in vitro model. Endogenous PLK1 and mTOR bound to each other in NCM460 cells (Figure 8A). Next, NCM460 cells were transfected with Si-PLK1 or pcDNA-PLK1 to investigate the effects of PLK1 on mTOR activity. The levels of p-mTOR and p-S6 tended to increase in the PLK1-deleted cells (Figure 8B). Moreover, overexpression of PLK1 abrogated the LPS-induced increases in the p-mTOR and p-S6 levels (Figure 8C). These findings imply that PLK1 negatively regulated mTOR activity in intestinal epithelial cells. Furthermore, the preincubation of NCM460 cells with rapamycin before LPS treatment elevated the expression of PLK1, and CLP mice treated with rapamycin also presented increased expression of PLK1 (Figure 9). The above results indicate a reciprocal regulation of PLK1 and mTOR in intestinal epithelial cells during sepsis.
Figure 8PLK1 physically interacts with mTOR and negatively regulates the mTOR pathway. NCM460 cells were selected as an in vitro model. A: PLK1 and mTOR physically interact with each other in NCM460 cells. B: NCM460 cells were transfected with Si-PLK1 or negative control siRNA and then exposed to lipopolysaccharide (LPS; 30 μg/mL) for 24 hours. The protein levels of p-mTOR, p-S6, and PLK1 were then measured. The graph shows the relative band densities. C: NCM460 cells were transfected with pCDNA3.1-PLK1 (a PLK1 knock-in plasmid) or control pCDNA3.1 plasmid (empty vector) and then exposed to LPS (30 μg/mL) for 24 hours. The protein levels of p-mTOR, p-S6, and PLK1 were then measured. The graph shows the relative band densities. Data are expressed as means ± SD. n = 3 independent experiments. ∗∗P < 0.01, ∗∗∗P < 0.001. IB, immunoblot; IP, immunoprecipitation.
Figure 9Rapamycin (Rapa) up-regulates PLK1 expression in the intestinal epithelium during sepsis. A: NCM460 cells were pretreated with rapamycin (50 nmol/L) for 5 hours and then exposed to lipopolysaccharide (LPS; 30 μg/mL) for 24 hours. The protein levels of p-mTOR, p-S6, and PLK1 were measured. The graph shows the relative band densities. B: The mice in the rapamycin group were intraperitoneally injected with rapamycin (10 mg/kg bodyweight) 1 hour after the cecal ligation and puncture (CLP) operation; the mice were then sacrificed 24 hours after CLP. The protein levels of p-mTOR, p-S6, and PLK1 in the intestine were measured. The graph shows the relative band densities. Data are expressed as means ± SD. n = 3 independent experiments. ∗∗∗P < 0.001. DMSO, dimethyl sulfoxide.
The present study indicated that enhanced autophagy resulting from rapamycin treatment reduces intestinal epithelial apoptosis, thus ameliorating sepsis-induced intestinal barrier dysfunction. Additionally, PLK1 protects against sepsis-induced intestinal barrier dysfunction by promoting intestinal epithelial autophagy in vivo and in vitro. Finally, the in vitro results indicated a reciprocal regulation of PLK1 and mTOR in LPS-treated intestinal epithelial cells (Figure 10).
Figure 10Diagram showing the role of the PLK1-mTOR axis in sepsis-induced intestinal barrier dysfunction. Overexpression of PLK1 enhances intestinal epithelial autophagy during sepsis, thus reducing intestinal epithelial apoptosis and ameliorating intestinal barrier dysfunction. However, the protective effects of PLK1 are impaired by treatment with chloroquine, an inhibitor of autophagy. PLK1 negatively regulates the activity of the mTOR pathway, and the inhibition of mTOR recovered the expression of PLK1, thus generating regulatory crosstalk during sepsis, which may be another mechanism of sepsis-induced intestinal barrier dysfunction. CLP, cecal ligation and puncture; LPS, lipopolysaccharide.
Autophagy has been demonstrated to play a critical role in enhancing intestinal barrier function in intestinal epithelial cells, which act as the first line of defense against the invasion of bacteria and isolate interactions of gut microbes with the host.
Furthermore, recent studies have shown that autophagy is connected with inflammatory responses in the intestine, although the pros and cons of autophagy are still controversial.
Inhibition of autophagic degradation process contributes to claudin-2 expression increase and epithelial tight junction dysfunction in TNF-alpha treated cell monolayers.
Activation of the EIF2AK4-EIF2A/eIF2alpha-ATF4 pathway triggers autophagy response to Crohn disease-associated adherent-invasive Escherichia coli infection.
In this study, the expression of autophagy-associated proteins was unchanged in the small intestines of septic rats, which is consistent with previous studies.
This phenomenon is also consistent with the findings that sepsis induced autophagy at early stages (8 hours post-CLP), which was weaker at later periods (from 12 to 24 hours after CLP).
The present study also illustrated that enhancing autophagy with rapamycin inhibited enterocyte apoptosis and restored the disrupted intestinal barrier, proving that autophagy plays a protective role in sepsis-induced intestinal barrier dysfunction.
The maintenance of normal intestinal barrier function is dependent on the balance between the proliferation and apoptosis of intestinal epithelial cells.
Notably, a previous study documented that sepsis induced excessive apoptosis of intestinal epithelial cells and thus increased intestinal mucosal permeability, ultimately leading to intestinal barrier dysfunction.
Apoptosis, a type of programed cell death, and autophagy, a highly conserved cellular recycling process, are two critical forms that regulate cell fate.
A previous study showed that autophagy deficiency in intestinal epithelial cells enhanced apoptosis induction under inflammatory conditions and thereby potentially decreased barrier integrity, consistent with the present results.
Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria.
The protective role of PLK1 in sepsis-induced intestinal barrier dysfunction was verified in previous studies, whereas the underlying mechanism is still being explored.
although little is known regarding its role in enterocyte autophagy during sepsis. Whether PLK1 induces or inhibits autophagy is still controversial. Some researchers have demonstrated that PLK1 inhibition induces different extents of autophagy, whereas others have shown that it attenuates autophagy.
Pharmacological inhibition of Polo-like kinase 1 (PLK1) by BI-2536 decreases the viability and survival of hamartin and tuberin deficient cells via induction of apoptosis and attenuation of autophagy.
The present study revealed that PLK1 overexpression promoted cell autophagy and preserved the intestinal barrier during sepsis in vivo and in vitro. Further study demonstrated that inhibiting autophagy with chloroquine impaired the rescued intestinal barrier and enterocyte apoptosis, whereas promoting autophagy with rapamycin reformed the intestinal barrier and enterocyte apoptosis in septic CAG-PLK1 mice, which verified the findings that PLK1 protects against sepsis-induced intestinal barrier dysfunction by promoting autophagy in intestinal epithelia.
The mTOR pathway is reported to play a critical role in regulating autophagy; phosphorylation of components in this pathway inhibits autophagy, and dephosphorylation of these components promotes autophagy.
In mammalian cells, mTORC1 phosphorylates autophagy-initiating UNC-5-like autophagy activating kinase 1 (ULK1), preventing the interaction and phosphorylation of ULK1 by adenosine monophosphate-activated protein kinase (AMPK), which is essential for ULK1 activation. ULK1/2 then phosphorylates autophagy-related gene 13 (ATG13) and FIP200, the two critical subunits of the ULK1/2 kinase complex. Thus, the initiation of autophagy by ULK is reciprocally regulated by mTORC1 and AMPK.
At the transcriptional level, mTORC1 phosphorylates transcription factor EB (TFEB), a master transcriptional regulator of lysosomal and autophagy genes, to prevent its nuclear translocation, thus inhibiting autophagy.
In this study, PLK1 was found to physically bind mTOR in NCM460 cells, providing the structural foundation of an interaction. Moreover, either down-regulation or overexpression of PLK1 negatively regulated the activity of the mTOR pathway in intestinal epithelial cells, consistent with earlier studies,
MiR-1224-5p activates autophagy, cell invasion and inhibits epithelial-to-mesenchymal transition in osteosarcoma cells by directly targeting PLK1 through PI3K/AKT/mTOR signaling pathway.
possibly explaining the promotive effect of PLK1 on autophagy. In addition, pretreatment of LPS-treated NCM460 cells with rapamycin also increased the expression of PLK1, indicating regulatory crosstalk between PLK1 and mTOR.
Limitations of this study, indicated next, were unavoidable. While it would be better to monitor enterocyte autophagy in different stages after CLP, the present study focused on sepsis-induced intestinal barrier dysfunction at 24 hours. The specific timepoint was selected based on a prior study demonstrating that the intestinal barrier deteriorated the most within 24 hours after CLP.
Second, the regulatory mechanism of PLK1 and mTOR in autophagy during sepsis remains unclear and is worthy of further investigation.
In summary, the study showed that intracellular intestinal PLK1 is required for protection of the intestinal barrier via promotion of autophagy and inhibition of apoptosis in intestinal epithelial cells during sepsis. Intestinal epithelial PLK1 mediated the suppression of sepsis-induced mTOR activation and thus regulates autophagy. These findings reveal that the reciprocal regulation of the PLK1-mTOR axis is crucial in sepsis-induced intestinal barrier dysfunction and might provide novel insight into enhancing autophagy for the treatment of sepsis.
Acknowledgment
We thank Professor Yu Zhang for her critical review of the manuscript.
Supplemental Data
Supplemental Figure S1Autophagic activity in the intestinal epithelium in cecal ligation and puncture (CLP) mice. Twenty-four hours after CLP, the mice were euthanized and analyzed. A: Representative images of LC3-II in the intestine in each group are shown. B: The levels of autophagy markers in the intestine were analyzed by Western blot. The graph shows the relative band densities. Data are expressed as means ± SD. n = 3 independent experiments. Scale bars = 100 μm.
Supplemental Figure S2PLK1 inhibition suppresses cell autophagy and deteriorates lipopolysaccharide (LPS)-induced apoptosis and high-permeability in vitro. Caco-2 cells were transfected with Si-PLK1 or negative control siRNA and then exposed to LPS (30 μg/mL) for 24 hours. A: The levels of autophagy markers in the intestine were analyzed by Western blot. The graph shows the relative band densities. B: The levels of apoptosis markers in the intestine in each group were measured. The graph shows the relative band densities. C: The levels of ZO-1 and Occludin in the intestine were analyzed by Western blot. The graph shows the relative band densities. D: Transepithelial electrical resistance (TEER) levels in each group. Data are expressed as means ± SD. n = 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council: Guide for the Care and Use of Laboratory Animals. Eighth Edition. National Academies Press,
Washington, DC2011
Inhibition of autophagic degradation process contributes to claudin-2 expression increase and epithelial tight junction dysfunction in TNF-alpha treated cell monolayers.
Activation of the EIF2AK4-EIF2A/eIF2alpha-ATF4 pathway triggers autophagy response to Crohn disease-associated adherent-invasive Escherichia coli infection.
Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria.
Pharmacological inhibition of Polo-like kinase 1 (PLK1) by BI-2536 decreases the viability and survival of hamartin and tuberin deficient cells via induction of apoptosis and attenuation of autophagy.
MiR-1224-5p activates autophagy, cell invasion and inhibits epithelial-to-mesenchymal transition in osteosarcoma cells by directly targeting PLK1 through PI3K/AKT/mTOR signaling pathway.
Supported by National Natural Science Foundation of China grant 82002092 (Y.-Y.C.), the Anhui Province Natural Science Foundation grant 2108085MH300 (W.-H.L.), the Natural Science Research Project of Colleges and Universities in Anhui Province grant 2021ZD0099 (Z.W.), and funding for the “Peak” Training Program for Scientific Research of Yijishan Hospital, Wannan Medical College, grants GF2019J03 and GF2019G08 (W.-H.L. and Y.-Y.C.).
Y.-Y.C., Y.Q., and Z.-H.W. contributed equally to this work.
The mechanisms underlying initiation and pathogenesis of idiopathic pulmonary fibrosis (IPF) via transforming growth factor beta 1 (TGF-β1) signaling are unclear. Using human and mouse fibroblasts and lung tissues and transgenic mouse models, Oruqaj and Karnati et al (Am J Pathol 2023, 259–274) explored these mechanisms. Peroxisomal biogenesis and metabolic proteins were significantly downregulated in all models in a Smad3-dependent manner. Targeting TGF-β1–Smad signaling axis may help manage IPF progression.
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