1 DSS - induced ulcerative colitis model established successfully
Mice quoted 5% DSS aqueous solution on the 2nd day began to appear unpleasant activity, body hair messy, loose stool and other symptoms; As the citation DSS extended, the DAI value raised, DAI mean of 3rd ,4th,5th and 6th day in homozygous group were 2.4, 4.2, 8.0 and 10.6. On the 6th day, more than half of the animals lost more than 15% of their initial weight, all the mice developed diarrhea or showed fecal occult blood. The naked eye can see the perianal shapeless blood stool attachment of male mice in homozygous group. Compared with the wild-type mice in control group, heterozygous mice developed symptoms after 4 days of exposure to DSS aqueous solution, DAI score also showed a certain upward trend, but the amplitude was not as significant as that of homozygous group, all the results above were shown in Fig. 1A.
2 Effects of colitis model on intestinal tissue morphology in Txnrd3 knockout mice
As shown in Fig. 1B, in the normal drinking water group, three genotypes of mouse colonic mucosal epithelium were intact, the epithelial cells were normal and closely arranged, the lamina propria intestinal glands were abundant, and plenty of goblet cells were found. In addition, lymphocyte infiltration was seen in homozygous group (green arrow). DSS treatment induced ulcerative colitis group mice colon can be seen obvious abnormal, wild group can be seen ulcer, mucosal layer can be seen ulcer (gray arrow), accompanied by a small number of lymphocytes and neutrophils infiltration (pink arrow); heterozygous mucosal layer can be seen ulcer (green arrow), accompanied by a small number of lymphocytes and neutrophils infiltration (red arrow), a small number of inflammatory cells infiltration into the submucosa, local intestinal gland dilatation (blue arrow); mice in homozygous group were further aggravated with large area ulcers (green arrows) in the mucosal layer, intestinal glands disappeared and replaced by proliferative connective tissue (yellow arrows), and some goblet cells were destroyed (blue arrows). A small number of red blood cells (gray arrows) and epithelial cells (black arrows) were found in the intestinal cavity. In NC group, the intestinal crypt depth of wild-type, heterozygous and homozygous mice were 0.1042, 0.11044, and 0.1298, respectively. Wild-type, heterozygous, homozygous mice in the UC group, the intestinal crypt depth was 0.21688, 0.0125786 and 0.2829, respectively.
3 Effects of colitis model on intestinal tissue ultrastructural structure in Txnrd3 knockout mice
In Fig. 1C, clear microvilli (red arrows) were observed in the colon of wild-type mice in the normal drinking water (NC) group by transmission electron microscopy. Cells structure was clear, neatly arranged and the number, size and morphology of organelles such as mitochondrial endoplasmic reticulum were normal. Heterozygous mouse colon cells were arranged neatly, with amount of microvilli reduced, number of mitochondria was regular, abnormal mitochondrial morphology (blue arrow) and occasional nuclear contraction (white arrow) was also observed. Homozygous mice colon tissue cells were randomly arranged and irregular, with abnormal mitochondrial morphology in slender strips, swelling and emptying of mitochondria, reduced fracture or disappearance of cristae (blue arrow), segregation in the pool of coarse endoplasmic reticulum (yellow arrow) and ER swelling (white arrow). DSS drinking water group wild-type mice colon tissue cell structure and boundaries are unclear, scattered, irregular, mitochondrial number increased deformation (blue arrow), individual cells approved deformation shrinkage (white arrow), endoplasmic reticulum swelling and pool isolation (red arrow); heterozygous colon tissue can be seen cell arrangement scattered nucleus deformation shrinkage, deep staining (green arrow), endoplasmic reticulum swelling (yellow arrow) and mitochondrial deformation (blue arrow). The colon structure of homozygous mice was irregular, the nucleus deformation wrinkle became smaller (green arrow), mitochondria swelling, ridge fracture or even disappearance (blue arrow), the number and volume of endoplasmic reticulum increased obviously, the morphology was swollen (yellow arrow), nucleus disappears and the organelles gradually dissolve into ruptured cells was seen (white arrow). In homozygote-UC group, purple arrows showed obvious rupture of cell membrane, cell contents flow out, swelling of mitochondria and endoplasmic reticulum which are regarded as iconic features of pyroptosis. In addition, transmission electron microscopy images showed subcellular features of necrotic-like swollen mitochondria.
4 Effects of colitis model on intestinal tissue immunohistochemical (IHC) in Txnrd3 knockout mice
In Fig. 2, IHC results of wild-type, heterozygous, and homozygous mouse colitis of ulcerative colitis model showed the expression level of IL-1β in the homozygous group colon increased significantly during ulcerative colitis compared with that in the wild group (p < 0.01); the expression level of c-Capase3 also increased in the homozygous group and the difference was significant (p < 0.05); the expression level of Caspase1 also increased significantly in the homozygous group and the difference was extremely significant (p < 0.01). In addition, there was no significant difference between heterozygous group and control group.
5 Relationship between Txnrd3 and cell death in CT26 cells
As shown in Fig. 3, CT26 cells transfected with Txnrd3 overexpression plasmids showed significant morphological changes and cell growth was inhibited or even died during the culture of colon cancer cells in CT26 mice. As Hoechst 33342 could penetrate the cell membrane, fluorescence of apoptotic cells after staining will be significantly enhanced than that of normal cells. Propidium iodide (PI) can not penetrate the cell membrane and can not be stained for normal or apoptotic cells with intact cell membranes. For necrotic cells, the integrity of their cell membranes was lost, and PI can stain necrotic cells. In Fig. 4, the proportion of positive cells PI staining increased significantly in Txnrd3 overexpression, but not in light blue apoptotic cells in Txnrd3 overexpression, while in Txnrd3 inhibition, positive cells in PI staining increased slightly, but no positive staining was found in the control group. A small number of positive staining cells were also found in the control group, considering that the transfection reagent was also toxic to the cells.
6 Calcium concentration in CT26 cells during Txnrd3 knockout / overexpression
Based on the same cell density in control, in Txnrd3 overexpression and Txnrd3 knock-down cells, the calcium content in the CT26 cells was significantly decreased in the Txnrd3 overexpression group than control group. As a result of endoplasmic reticulum stress increase, Txnrd3 overexpression can cause intracellular calcium leakage while presented a slight increase of calcium in Txnrd3 knockout group as shown in Fig. 4A.
7 Oxidative stress level in CT26 cells during Txnrd3 knockout / overexpression
In Fig. 4B, the results of ROS staining positive CT26 cells in the Txnrd3 overexpression group increased significantly compared with control group, while the proportion of green fluorescent cells in the Txnrd3 inhibition group increased slightly compared at the same time, but it was not significant as overexpressed. Considering Txnrd3 inhibition can also influence oxidative stress in CT26 cells.
8 Effects of Txnrd3 on mRNA expression of necrosis and pyroptosis pathway genes in colonies and CT26 cells
In the transfected CT26 cells, the expression levels of NLRP3, ASC, Caspase1, IL-1β and IL-18 of pyrolytic-related genes in the Txnrd3 overexpression group were significantly higher than those in the control group with the difference especially significant (p < 0.01). The expression level of GSDMD increased significantly (p < 0.05); the expression of necrotizing pathway related gene MLKL, RIPK3 increased extremely significant (p < 0.01) in Txnrd3 overexpression group, however, the RIPK1 has no significant changes. The expression of apoptosis-related genes Bax, Caspase8 and Caspase3 was significantly increased (p < 0.01), and Bcl2 expression decreased significantly (p < 0.05). In the Txnrd3 inhibition group, expression of focal death gene NLRP3, ASC, IL-1β decreased significantly, and the difference is extremely significant (p < 0.01); GSDMD expression was significantly decreased (p < 0.05); RIPK3 expression significantly decreased (p < 0.01). In addition, expression of apoptosis-related gene Bax, Caspase8 increased significantly, while expression of Caspase3 and Bcl2 decreased at the same time (p < 0.05).
9 Detection of necrosis and pyroptosis pathway proteins in colonies and CT26 cells
As shown in Fig. 6A-B, in transfected CT26 cells the protein expression levels of GSDMD, RIPK3, MLKL, NLRP3, Caspase1 and IL-1β in the Txnrd3 overexpression group were significantly higher than those in control group with the difference especial significant (p < 0.01). Expression of Capase1 and MLKL was differentially decreased in Txnrd3 knockdown group (p < 0.01), but NLRP3 expression was increased at the same time (p < 0.01). Results indicated that Txnrd3 overexpression could lead to necrosis and pyroptosis in CT26 cells. In addition, homozygote-NC group relative protein expression in colons was differentially increased compared with wild-NC group (p < 0.01); expression of GSDMD, Caspase1, NLRP3, RIPK3, MLKL was up-regulated in wild-UC group (p < 0.01); GSDMD, Caspase1, NLRP3, RIPK3, MLKL was most differentially expressed in homozygote-UC group compared with wild-NC and wild-UC group (p < 0.01).
10 Effects of Txnrd3 on oxidative stress in colon and Txnrd3 transfected CT26 cells
Which can be observed from Fig. 6C-D, antioxidant activity of transfected CT26 cells results showed that the SOD activity and T-AOC activity of Txnrd3 overexpression group differentially decreased 33.4% and 65.3% with significant difference (p < 0.01); with MDA content increased to 2.98 times (p < 0.01), the T-AOC activity in the Txnrd3 knockout group decreased 24.6%, MDA content increased 87.6%, SOD activity showed a downward state but the difference was not significant (p > 0.05). In vitro results of oxidative stress suggest that SOD activity and T-AOC activity of homozygote-NC group was differentially decreased 25.3% and 21.6% with significant difference compared with wild-NC group (p < 0.01), with MDA content increased to 1.4 times (p < 0.01). SOD activity and T-AOC activity of homozygote-UC group colon tissue decreased 41.6% and 47.9% respectively, with MDA content increased to 1.97 times (p < 0.01).