| Objective: To observe the effect and anti-apoptotic mechanism of Shaoyao Decoction’s intervention in mice with radioactive intestinal injury.By observing the general condition of mice at various time points following ionizing radiation,the histopathological morphology of the mouse intestine,the level of antioxidant and antiinflammatory indexes in intestinal tissues by ELISA,the apoptosis of colonic cells by TUNEL,the response of Dclk1/ATM to DNA damage by Western blot,q RT-PCR,and immunofluorescence staining were used to investigate the possible signaling mechanism of inhibition of apoptosis of intestinal mucosal cells by Shaoyao Decoction.To understand the unique mechanism of action of Shaoyao Decoction in the treatment of radioactive intestine damage at the overall,cellular,and molecular levels,as well as to offer an empirical foundation for Shaoyao Decoction’s therapeutic application.Methods: 1.48 SPF male C57BL/6 mice were randomly divided into 6 groups(n=8):blank group,model group,positive drug group,and low,medium and high dose groups of Shaoyao Decoction.48 healthy male C57BL/6 mice were randomly assigned to one of six groups(n=8): a blank group,a model group,a positive drug group,and low,medium,and high Shaoyao Decoction dosage groups.With the exception of the blank group,all mice were sedated intraperitoneally with sodium pentobarbital(20 mg/kg),followed by a one-time abdominal 6MV-X-ray irradiation in a Swiss Medical Tech Precise linear accelerator to create a mouse model of radioactive intestine damage.2.Ground 107 g Shaoyao Decoction mixture consisting of 30 g peony,15 g scutellaria,15 g coptis chinensis,9g rhubarb,6g xylobacter,6g areca,15 g angelica,5g cinnamon and 6g licorice,and obtain 15 g lyophilized peony soup powder after treatment.The concentrations of high,medium and low doses of lyophilized powder of Shaoyao Decoction were set at 0.36g/m L,0.24g/m L and 0.12g/m L with distilled water.The dosages of Shaoyao decoction in the three groups were 8.56g/kg,17.12g/kg and 25.68g/kg(raw herbs),respectively.3.Therapeutic administration began 24 hours after modeling and continued twice daily at 12 hour intervals for 7 days.The blank group received no intervention,the model group received saline 0.2 m L by gavage,the positive drug group received dexamethasone 0.12 mg/kg/d by gavage,and the Shaoyao Decoction high,medium,and low dose groups received 0.36 g/m L,0.24 g/m L,and 0.12 g/m L of Shaoyao Decoction’ lyophilized powder,respectively.4.Observe the general condition of the mice in each group,using HE staining and Masson staining of intestinal histopathological morphology,and(ELISA)measured the expression of MDA,SOD,COX,LPS,IL-6,IL-1β,and TNF-α factors to determine the radiological intestinal injury associated with oxidative stress and inflammatory response.5.TUNEL was used to detect apoptosis in colon tissue cells,and Western blot and q RT-PCR were used to detect the expression of ATM,Dclk-1,MRE-11,Bcl-2,Bax,Caspase-3,and Cyto-C proteins and genes;immunofluorescence labeling was used to evaluate the expression levels of p53 and Claudin-1 proteins.6.Statistical Analysis: Excel and Graph Pad Prism 8 software were used for graphing and statistical analysis,and data were represented as mean standard deviation(X±s)using one-way ANOVA;Masson staining was quantified using Image-Pro Plus 6.0 software,and Western blot bands were quantified using Image J software.Statistically significant differences were defined as P < 0.05 or P < 0.01.Results: 1.General condition: After modeling,mice in each group showed reduced activity,loss of fur without luster,and decreased feeding and drinking with pus and blood feces when compared to the blank group.The aforesaid symptoms and indicators improved in the treated group following drug administration compared to the model group,and the model group’s body weight was dramatically lowered compared to the control group.The model group had a statistically significant drop in body weight compared to the blank group(P < 0.05),while the Shaoyao Decoction high-dose group and dexamethasone group had a statistically significant rise in body mass compared to the model group(P < 0.05).2.Histopathological morphology of intestine: HE staining results suggest:The blank group’s intestine was structurally intact,with relatively intact villi structure,abundant glands,mild intestinal inflammation,and no damage to the mucosal layer crypt or other pathological injury changes;the model group’s intestine was structurally damaged,with damage to the mucosal layer crypt,fibrous tissue proliferation,and epithelial cell detachment;the dexamethasone group’s intestine showed local slight epithelial cell necrosis.Masson staining results suggest: The collagen fibers in the model group were clearly blue compared with the blank group;The spectrophotometric optical densities of the model component was significantly higher than that of the blank group,which was statistically significant(P < 0.01),and the spectrophotometric optical densities of the Shaoyao Decoction group and dexamethasone group was lower than that of the model group,which was statistically significant(P < 0.01).3.The levels of antioxidant and anti-inflammatory indexes were detected by ELISA: the SOD activity in serum was decreased(P < 0.01)and the MDA content was increased(P < 0.05)in the model group compared with the blank group,and the SOD activity in serum was increased and the expression of MDA was decreased(P <0.01)in the high,medium and low dose groups of Shaoyao Decoction,the control group,and the model group.The levels of COX,LPS,IL-6,IL-1β and TNF-α in serum were increased in the model group compared with the blank group(P < 0.01);the levels of COX,IL-6,IL-1β and TNF-α in serum were decreased in the high,medium and low dose groups of Shaoyao Decoction and the dexamethasone group compared with the model group(P < 0.01);the levels of LPS in serum were decreased in the low dose group of Shaoyao Decoction compared with the model group(P < 0.05),while the effect was more significant in the high and medium-dose groups(P < 0.01).4.The apoptosis level of mucosal epithelial cells in colon tissue: compared with the blank group,the model group showed a stronger positive TUNEL immunofluorescence signal,and the number of positive cells was significantly higher than the blank group and was statistically significant(P < 0.01).Compared with the model group,the number of positive TUNEL cells in the dexamethasone group,Shaoyao Decoction high,medium,and low dose groups was significantly lower and statistically significant(P < 0.01),in which the immunofluorescence signal of each group of Shaoyao Decoction decreased with increasing dose,and the most obvious in the high dose group of Shaoyao Decoction.5.The expression levels of ATM,Dclk-1,MRE-11,Bcl-2,Bax,Caspase-3,Cyto-C-related proteins and genes were detected by Western blot method and q RTPCR: the gene and protein levels of ATM,Dclk-1 and MRE-11 in the colon of mice in the model group were significantly reduced compared with those in the control group.The expression of mitochondrial pro-apoptotic genes and proteins Bax,Caspase-3 and Cyto-c was significantly increased in the model group(P < 0.01),while the expression of anti-apoptotic genes and protein Bcl-2 was decreased.ATM,Dclk-1,MRE-11,and Bcl-2 were significantly increased and Bax,Caspase-3,and Cyto-c were significantly decreased after treatment with Shaoyao Decoction.6.Immunofluorescence staining detected the protein expression levels of p53 and Claudin-1 in colonic tissues: compared with the blank group,the model group significantly increased the expression of p53 and significantly decreased the level of Claudin-1 after radiation irradiation(P < 0.05).And Shaoyao Decoction treatment decreased the level of p53 and Claudin-1 expression was increased(P <0.05).Conclusions:Shaoyou Decoction may promote the expression of Dclk1 in Tuft cells of crypt and promote the rapid activation of DDR competing p53 apoptosis pathway by ATM,meanwhile,exert paracrine effect to promote the expression of intestinal mucosal repair factor Claudin-1 and accelerate the reconstruction of crypt structure,thus interfering with radioactive intestinal injury. |
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