ABSTRACT
Objective:To explore the efficacy and molecular mechanism of Zhizhuwan decoction and its ingredient-contained serums on the proliferation and apoptosis of rat colon interstitial cells of cajal (ICC), and make a molecule-level analysis of the possible mechanism of traditional Chinese medicine (TCM) purgation-tonifying therapy in treating slow transit constipation (STC). Method:A total of 40 rats were divided into Atractylodis Macrocephalae Rhizoma(AMR) group, Aurantii Fructus Immaturus（AFI） group, Zhizhuwan group and blank serum group on random basis, with 10 in each group. Baizhu group was given 17.7 g·kg-1·d-1 of AMR decoction by gavage, AFI group was given 8.9 g·kg-1·d-1 AFI decoction by gavage, Zhizhuwan group was given 26.4 g·kg-1·d-1 Zhizhuwan decoction by gavage, and blank serum group was given 3 mL sterile distilled water for 7 consecutive days, once a day. Drug-contained serums and blank serum were collected from blood of the above groups and diluted to 5%, 10%, 15% and 20% concentrations. Each concentration was intervened for 24 h and 48 h, and the amount and status of ICC were observed. The best intervening concentration and time for each group with cell counting kit-8 (CCK-8) were determined. Rat colon ICC was divided into blank control group, blank serum group, AMR group, AFI group and Zhizhuwan group. ICC proliferation for each group was detected with EdU, ICC apoptosis for each group was detected by flow cytometry, and expressions of X-linked inhibitor of apoptosis protein (XIAP) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. Result:Compared with the normal group, the best intervention concentration for blank serum group, AMR group and AFI group was 10%, while that for Zhizhuwan group was 5%. The best intervention times for the above groups were all 24 h. No distinct difference between the effect of blank control group and blank serum group on the proliferation and apoptosis of ICC was observed. In comparison with blank control group and blank serum group, AMR group, AFI group and Zhizhuwan group showed significant changes in ICC proliferation rate (P<0.05，P<0.01). There was a greater increase in ICC proliferation rate of Zhizhuwan group than that of AMR group and Zhizhu group (P<0.05，P<0.01), with no distinct difference between the changes of ICC proliferation rates in AMR group and AFI group. There was no significant difference between the changes of ICC apoptosis rates in AMR group, AFI group and Zhizhuwan group than in blank control group and blank serum group. There were significant increases in the expressions of XIAP and PCNA in AMR group, AFI group and Zhizhuwan group than in blank control group and blank serum group (P<0.05，P<0.01)， but with little difference among the three groups. Conclusion:At certain concentrations, Zhizhuwan, AFI and AMR all have the effect in improving ICC proliferation by increasing XIAP and PCNA expressions, with no evident effect on the apoptosis of ICC, based on TCM purgation-tonifying therapy, Zhizhuwan has the effect in improving ICC proliferation, with a better effect than single administration with AFI or AMR.
ABSTRACT
Objective:To explore the effect of Zhizhuwan on intestinal conduction and expressions of Phospholipase C-γ1 (PLC-γ1)/Phospholipase C-γ2 (PLC-γ2) signaling pathway of slow transit constipation (STC) with spleen deficiency syndrome. Method:Special pathogen free (SPF) healthy mice were randomly divided into normal group and model making group. Folium Sennae gavage was used to induce the spleen deficiency status, and then diet and drinking water were controlled to establish the mice model of spleen deficiency constipation. After the modeling, the mice in modelling group were randomly divided into model group, Zhizhuwan group and mosapride group. Zhizhuwan group was given drug at the dose of 9.0 g·kg-1·d-1, mosapride group was given 2.5 mg·kg-1·d-1, model group and normal group were given the equal dose of distilled water for 7 consecutive days. At the end of the treatment, the length of Indian ink in the colon was used to calculate the intestinal propulsion rate of the mice. The D-xylose kit was used to determine the content of D-xylose in serum of mice. The hematoxylin eosin (HE) staining was used to observe the pathological changes of colon tissues in mice. The immunohistochemistry and Western blot were used to detect the expression levels of PLC-γ1 and PLC-γ2 proteins in colon tissues of mice. Real-time PCR was used to detect the mRNA expression levels of PLC-γ1 and PLC-γ2. Result:Compared with normal group, the intestinal propulsive rate and the serum D-xylose value in model group were significantly decreased (Pγ1 and PLC-γ2 in colon tissues were significantly decreased (PD-xylose in Zhizhuwan group and mosapride group were significantly increased (Pγ1 and PLC-γ2 were significantly increased (PConclusion:Zhizhuwan can promote the intestinal movement in slow transit constipation model mouse with spleen deficiency syndrome, and alleviate the symptoms of constipation in mice. The related mechanism may be related to the increase of the expressions of PLC-γ1 and PLC-γ2 in colon tissues of mice with spleen deficiency and constipation.