| Objective:Shenling Baizhu Powder which has the effect of invigorating the spleen qi and infiltrating dampness has certain curative effects on ulcerative colitis,Crohn's disease,and diarrhea-predominant irritable bowel syndrome.Previous studies have shown that this prescription 's effect on intestinal diseases is related to the protection of intestinal barrier function.This experiment is to explore the mechanism of injury of the intestinal epithelial barrier and to find out whether the effect of Shenling Baizhu powder on UC is related to the regulation of intestinal tight junction and goblet cells by establishing UC mouse model,which will provide a scientific basis for the clinical application of Spleen-invigorating prescriptions for the treatment of UC.MethodsSixty male SPF C57 BL / 6 mice weighing 18-22 g were divided into normal group(n = 10)and model group(n = 60)according to random number table.The model group suffered colonic injury by drinking 3% sodium dextran sulfate(DSS molecular weight 9,000-2,0000 units)for six days.The mouse's mental status ?weight?stool characteristics and blood in the stool were observed and recorded daily.After modeling,the mouse were numbered according to body weight and divided into model group,sulfasalazine group(SASP),high?medium and low dose of Shenling Baizhu Powder group according to random number table.Each group consists of ten mouse.The mouse in the blank group and the model group were given 0.9% saline by 0.2ml / 10 g,while the positive group was given 0.52 g /(kg · d)SASP tablet solution.The mouse in the Shenling Baizhu Powder group were given high(1.56 g / ml),medium(0.78 g / ml),low(0.39 g / ml)dose of concentrated decoction orally,once daily for a total of 14 days.Materials and testing: After the last administration,the mouse in each group were fasted for 24 h,then,they were anesthetized with 10% chloral hydrate by 0.1ml/10 g.Blood was collectsd by eyeball-taking method.The supernatant was collectsd after the blood was centrifuged and then used for ELISA detection of cytokines IL-1?.After blood sampling,the animals were sacrificed and the abdominal cavity was dissected.The whole colon was cut out and the length of the colon was measured,then the net weight of the colon was weighed after the colon contents were washed out.About 1cm lesion colon was intercepted,then,fixed with 4% paraformaldehyde for HE staining and AB / PAS staining.The pathological changes of colonic tissue of mouse in each group were observed,and mucosal inflammation score and number of goblet cells in colon tissue were recorded.The remaining colon tissues were placed in non-enzyme cryopreservation tube and stored in a refrigerator at-80 ? for subsequent western blot detection of protein expression and ELISA detection of colonic cytokine levels.Results1.Mouse's general condition and DAI score after modeling: The mouse in each group nearly had the same body weight before modeling,with shiny coat,sensitive activity.Their stool was normal,and the occult blood test was negative or weakly positive.At the beginning of modeling,the weight of mice in the model group gradually decreased,and even lower than the mean value at the beginning of adaptive feeding.Compared with the normal group,the difference was significant(P<0.01).Their stools gradually thinning and the occult blood stools developed into bloody stools.The DAI score was significantly increased(P <0.001).2.General conditions,DAI scores,and pathological changes of the mouse after administration: During the administration period,all mouse in the control group were normal;the spirit status of the mouse in the model group gradually improved,their dietary amount and body weight also increased,and the bloody stool decreased;With the prolongation of the administration time,the body weight of the mouse in the treatment groups increased gradually,especially that in the sulfasalazine group and the high-dose Shenling Baizhu Powder group obviously increased,but there was no statistically significant difference compared with model group(P>0.05).At the end of administration,there was no significant difference in body weight between the model group and other foue administration groups(P>0.05).Compared with the control group,the length of unit colon in the model group was significantly shorted(P <0.01),The unit colon weight was significantly increased(P<0.01).Compared with the model group,there was an increasing trend in the length of colon in each administration group,but there was no statistical significance(P<0.05);The weight of colon in each administration group decreased,but there was no significant difference(P <0.05).HE staining showed that in the normal mouse,the colon walls were all intact,the mucosal microvilli were arranged neatly,the epithelial cells were arranged regularly,and the crypt structure was normal.In the model group,the mucous layers of the colon of the mouse were damaged and shedding,and the cells were disordered.Some of the crypts disappeared and part of them expanded.The distribution of the glands was irregular and shortened.The submucosa layer was sparse,and a large number of eosinophils infiltrated.Less pathological changes were shown in the muscle layer.The intestinal inflammation score was significantly higher than that of the normal group(P<0.001).In the sulfasalazine group,the microvilli? crypts and glands of the colonic mucosa were slightly shorter than those in the normal group.The arrangement of the epithelial cells was slightly disordered but there was no shedding.A small part of the crypts dilated,the inflammatory cells in the submucosa were reduced,and the muscle layer had no lesions.The major improvement in the pathological changes of the colon in the low dose group compared with the model group was that the mucosal layer was intact and the epithelial cells did not appear to fall off.In the medium and high dose groups,the microvilli and crypts of the colon mucosa and the gland ducts were also shorter than those in the normal group,but the mucosal layers were complete,a small part of the crypts expanded with neutrophils infiltrated,and the submucosa in the middle dose group was loose,with a small amount of inflammatory cells.But this pathological phenomenon were significantly improved in the high dose group.Cooper scores of these two groups were decreased.3.The translational expression of NLRP3 and NLRP6 in the colon,the levels of serum IL-1? and IL-18 in tissues,the number of colonic goblet cells : The expression of NLRP3 protein in colon tissue of mice in model group was significantly higher than that in normal group(P<0.01).Both the sulfasalazine group and the high dose of Shenling Baizhu Powder group could significantly down-regulate the expression of NLRP3.Especially in the high-dose of Shenling Baizhu Powder group,NLRP3 was almost reduced to the normal group level,and there was a significant difference compared with the model group(P<0.01).However,no significant difference was shown between the medium or low doses of Shenling Baizhu Powder group and the model group(P>0.05).The level of serum IL-1? in the model group increased by about 1/3 compared with the normal group,which was statistically significant(P<0.05).Both SASP and high doses of Shenling Baizhu Powder can reduce the concentration of IL-1? in serum.Compared with the model group,the difference was statistically significant(P<0.05).The concentration of IL-1? of middle and low doses of Shenling Baizhu Powder was only slightly lower than that of the model group,and the difference was not significant(P>0.05).There was no significant difference in the level of IL-18 in colon tissues among each group(P>0.05).The expression of NLRP6 protain and the number of goblet cells in the colon of mice in the model group were significantly lower than those in the control group(P<0.01).The expression of NLRP6 in the sulfasalazine group was significantly higher than that in the model group(P<0.001).The expression of NLRP6 in each dose of Shenling Baizhu Powder group was also significantly higher than that in the model group(P<O.O5);The number of goblet cells in the colonic epithelium of the mice in the sulfasalazine group was significantly higher than that of the model group(P<0.001)and that in the high-dose Shenling baizhu Powder group was also significantly increased,compared with the model group,there was a statistical difference(P<0.05).The number of goblet cells in the colonic epithelium of mice in the Shenling Baizhu Powder group was lower than that in the model group,with no statistical significance(P>0.05).4.The translational expression of P-65?MLCK?P-MLC and Occludin in colon:The expression of occluding protein in colonic tissue was significantly lower in the model group than that in the control group(P <0.05),and the expression of occludin protein in high dose Shenling Baizhu Powder group was significantly higher than that in the model group(P<0.05).The translational expression of P-65 in the model group was increased to a great extend(P<0.01),while it was effectively reduced in sulfasalazine and high doses of Shenling Baizhu Powder groups(P<0.01 or P<0.05).The expression of MLCK protein in the colon of the model group was significantly increased(P<0.01),while it was effectively reduced in each administration group(P<0.01).The MLC expression in each group is almost the same(P>0.05),but the expression of P-MLC in the model group was surged(p<0.01),and decreased in the sulfasalazine group,which was close to the normal group(p<0.01).The P-MLC expression in the medium and high dose of Shenling Baizhu Powder groups were also significantly down-regulated(P<0.05).Conclusions1.After administering Shenling Baizhu Powder,the general condition of the mice was significantly better than that of the model group,and the inflammation of the colonic mucosa was relieved.This indicated that the Shenling Baizhu Powder could promote the recovery of the UC mice to some extent,and improved the intestinal mucosal pathological injury.2.After administration of Shenling Baizhu Powder,the colonic NLRP3 expression and serum IL-1? levels were significantly down-regulated,suggesting that Shenling Baizhu Powder may reduce intestinal inflammation by down-regulating intestinal NLRP3 expression and decreasing the level of active IL-1?.So as to relieve intestinal mucosal damage.3.After administration of Shenling Baizhu Powder,the expression of NLRP6 in colon of mice was increased compared with the model group,and the number of goblet cells was also increased.The effect of Shenling Baizhu Powder on the repair of intestinal mucosa may be related to the promotion of goblet cell proliferation.4.After administration of Shenling Baizhu Powder,the expression of occludin protein in the colon of mice was increased,the activation of MLCK was inhibited,and the phosphorylation of MLC was reduced.This indicated that Shenling Baizhu Powder may regulate intestinal tight junction proteins by inhibiting the activation of MLCK/MLC pathway.As a result to maintain the normal permeability of the intestinal mucosa. |
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