| Objective To investigate the intervention effect of total saponins of Panax japonicus on excessivel proliferation of colon stem cells in mice fed with high fat diet and its molecular mechanism.Background With the change of dietary structure in human society,high-fat diet promotes the excessive proliferation of Intestinal stem cells and increases the risk of colorectal cancer,which is one of the most lethal malignant tumor diseases in the world.Methods 1)32 SPF 8-week-old C57BL/6 mice were randomly divided into normal chow diet group(n = 8),high-fat diet group(n = 8)and low-dose and high-dose groups of total saponins of Radix Scutellariae(n = 8).The normal chow diet group was given routine diet maintenance,the high-fat diet group and the low-dose group were given high-fat diet(see the animal experimental method on page 6 of the text for details of the high-fat diet).The drug group was given intragastric administration of total saponins of 50mg/kg and 150mg/kg every day,and the remaining two groups were given corresponding doses of 0.5% carboxymethyl cellulose sodium intragastric administration,six days a week,and no medicine on Sunday.The mice were continuously fed for 4 months.After fasting for 24 hours,the mice were killed and fresh colon tissue was frozen or embedded in paraffin.2)HE staining was used to observe the morphological changes of colonic epithelium in mice.3)Immunohistochemical staining was used to detect the proliferation index of mouse colonic stem cells: the protein expression levels of PCNA,intestinal stem cell marker SOX9 and Wnt pathway related proteins: GSK-3βand β-catenin.4)Western blot method was used to detect the related indexes of apoptosis in mouse colonic tissue: the protein expression levels of Caspase12,Caspase9 and Caspase3.5)The expression of endoplasmic reticulum stress-related proteins in mouse colon was detected by Western blot method: GRP78,p-PERK,p-EIF2α,ATF4,Chop,ATF6,p-IRE1,XBP1.Results 1)HE staining showed that the morphology of epithelium in the high-fat diet group was still intact and the crypt depth was longer than that in the normal chow diet group,and the colon crypt depth in the low-dose and high-dose group was lower than that in the high-fat diet group(P<0.05).2)Immunohistochemical results showed that compared with the normal chow diet group,the expression of PCNA and stem cell marker SOX9 protein in colon of high fat diet group was significantly increased(P<0.001).The expression of GSK-3β,a protein related to Wnt pathway,was significantly decreased,and the expression of β-catenin was significantly increased(P<0.01).Compared with the high fat diet group,the expression of PCNA and SOX9 protein decreased,the expression of GSK-3β increased and the expression of β-catenin decreased in the treatment group(P<0.05,P<0.01 or P<0.001).3)the results of Western blot showed that: compared with the normal chow diet group,the colon apoptosis-related protein in the high-fat diet group was significantly down-regulated(P<0.05 or P<0.01).After the intervention of low and high dose of total saponins of Radix Scutellariae,the level of apoptosis-related protein increased,among which Caspase12 and Caspase3 were statistically significant(P<0.05 or P<0.01).4)the results of Western blot showed that compared with the normal chow diet group,the expression of endoplasmic reticulum stress-related proteins GRP78,p-PERK,p-EIF2α,ATF4,CHOP,ATF6,p-IRE1 and XBP1 in the high-fat diet group was down-regulated(P<0.05,0.01 or P<0.001).The expression of endoplasmic reticulum stress-related proteins was up-regulated after intervention with low and high dose of total saponins of Radix Scutellariae(P<0.05,0.01 or P<0.001).Conclusion Total saponins of Panax japonicus may inhibit the excessive proliferation of colonic stem cells and maintain intestinal homeostasis by down-regulating Wnt/β-catenin signal pathway and inducing apoptosis of colonic endoplasmic reticulum stress-dependent epithelial cells in mice with high-fat diet. |
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