Objective

2

To explore the possible mechanism of

Zhizhuwan Decoction

（枳术丸汤剂， ZD） for treatment of slow transit constipation （STC）.

Methods

2

Twenty SD male rats were randomized into ZD group and blank group， with 10 rats in each group. The rats in the ZD group were given 5.8 g ZD by gavage at a concentration of 2 g/ml per day for seven consecutive days， while those in the blank group were fed normally. One hour after the last administration， blood was collected from the abdominal aorta and centrifuged to prepare the medicated serum. CCK8 method was used to assess the cell inhibition rate of U73122， a specific inhibitor of phospholipase C-γ （PLC-γ），on primary interstitial cells of Cajal （ICC）； the concentration of medicated serum was selected from 5%， 10%， 15% and 20% by observing the effect on cell proliferation of ICC； the medication time was selected from 12 h， 24 h and 48 h based on the results. Second or third generation of ICC were seeded into a 96-well plate， with 1×10

4

cells per well and three repeated wells for each of the three groups， which were normal group， model group （normal cells plus U73122 at the selected concentration）， blank serum group （model group plus 10% blank serum） and ZD-containing serum group （model group plus ZD-containing serum at the selected concentration）； the administrations lasted for the selected time duration. The positive expression of PLC-γ 1 and PLC-γ 2 were detected by immunohistochemistry， and the protein and gene expression of PLC-γ 1 and PLC-γ 2 were detected through Western blotting and RT-PCR， respectively.

Results

2

The selected best concentration was 30 μmol/L of U73122， and was 5% of ZD； and the selected administration time was 24 h. Compared to the normal group， the model group had decreased PLC-γ1， PLC-γ2 average optical density， protein and gene expression （

P

＜0.01）； compared to the model group， the ZD-containing serum group and blank serum group had higher PLC-γ1 and PLC-γ2 average optical density， protein and gene expression （

P

＜0.05 or

P

＜0.01）； compared to the blank serum group， the ZD-containing serum group had significantly increased positive expression of PLC-γ1， PLC-γ2， as well as protein and gene expression （

P

＜0.01）.

Conclusion

2

ZD may regulate cell proliferation through activation of the PLC-γ signaling pathway of ICC， so as to promote the gastrointestinal motility.