Journal of Traditional Chinese Medicine ›› 2023, Vol. 43 ›› Issue (6): 1160-1167.DOI: 10.19852/j.cnki.jtcm.2023.06.008
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Hetong decoction (和通汤) relieves loperamide-induced constipation in rats by regulating expression of aquaporins
HUANG Xi1, XIA Youguang1, HAN Kai1, QI Weiwei1, LINGHU Qing1, ZHANG Qing2, XUE Jingbo1(
)
- 1 Department of Anorectal, Xuzhou Hospital Affiliated to Nanjing University of Chinese Medicine, Xuzhou 221000, China
2 Department of Pathology, Xuzhou Hospital Affiliated to Nanjing University of Chinese Medicine, Xuzhou 221000, China
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Received:2021-05-28Accepted:2021-08-25Online:2023-10-25Published:2023-11-01 -
Contact:XUE Jingbo, Department of Anorectal, Xuzhou Hospital Affiliated to Nanjing University of Chinese Medicine, Xuzhou 221000, China. jingboxue20@126.com. Telephone: +86-15005202965
Cite this article
HUANG Xi, XIA Youguang, HAN Kai, QI Weiwei, LINGHU Qing, ZHANG Qing, XUE Jingbo. Hetong decoction (和通汤) relieves loperamide-induced constipation in rats by regulating expression of aquaporins[J]. Journal of Traditional Chinese Medicine, 2023, 43(6): 1160-1167.
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Table 1 Primer information and primer sequences used for the quantitative real-time PCR analysis
| Gene | Forward | Reverse |
|---|---|---|
| Aquaporin 3 | 5’-CTTCTTTGACCAGGACCGGC-3’ | 5’-TACACGAAGACACCCGCAAT-3’ |
| Aquaporin 8 | 5’-AGAGTCCGATGTTTGTGCCA-3’ | 5’-AGTACGGGAGGAGCATCAC-3’ |
| Glyceraldehyde-3-phosphate dehydrogenase | 5’-GCAACCGGGAAGGAAATGAATG-3’ | 5’-CCCAATA-CGACCAAATCAGAGA-3’ |
Table 1 Primer information and primer sequences used for the quantitative real-time PCR analysis
| Gene | Forward | Reverse |
|---|---|---|
| Aquaporin 3 | 5’-CTTCTTTGACCAGGACCGGC-3’ | 5’-TACACGAAGACACCCGCAAT-3’ |
| Aquaporin 8 | 5’-AGAGTCCGATGTTTGTGCCA-3’ | 5’-AGTACGGGAGGAGCATCAC-3’ |
| Glyceraldehyde-3-phosphate dehydrogenase | 5’-GCAACCGGGAAGGAAATGAATG-3’ | 5’-CCCAATA-CGACCAAATCAGAGA-3’ |
Figure 1 Effect of HTT on the defecation function in rats with loperamide-induced constipation A, B: the total fecal weight within 24h and fecal water content of rats in different groups were measured every week since the first week of HTT or mosapride administration. C, D: rats in different groups were intragastrically administrated with the activated carbon suspension and sacrificed after 30 min for calculation of the relative transit ratio (activated charcoal travel distance/full length of small intestine × 100%). Control group was administered intragastrically with equal amount of 0.9% sodium. Model group was administered with normal saline. HTT-low (L), HTT-medium (M), HTT-high (H) groups were intragastrically given 95 g/kg HTT (HTT-L), 190 g/kg HTT (HTT-M) or 380 g/kg HTT (HTT-H) twice a day for consecutive 30 d. Positive control group was given 1.60 mg/kg mosapride (MedChemExpress) lavage once every two weeks for 4 weeks. HTT: Hetong decoction. Data were expressed as mean±standard deviation (n = 10). aP < 0.001 vs Control. cP < 0.05, dP < 0.01 and bP < 0.001 vs Model.
Figure 1 Effect of HTT on the defecation function in rats with loperamide-induced constipation A, B: the total fecal weight within 24h and fecal water content of rats in different groups were measured every week since the first week of HTT or mosapride administration. C, D: rats in different groups were intragastrically administrated with the activated carbon suspension and sacrificed after 30 min for calculation of the relative transit ratio (activated charcoal travel distance/full length of small intestine × 100%). Control group was administered intragastrically with equal amount of 0.9% sodium. Model group was administered with normal saline. HTT-low (L), HTT-medium (M), HTT-high (H) groups were intragastrically given 95 g/kg HTT (HTT-L), 190 g/kg HTT (HTT-M) or 380 g/kg HTT (HTT-H) twice a day for consecutive 30 d. Positive control group was given 1.60 mg/kg mosapride (MedChemExpress) lavage once every two weeks for 4 weeks. HTT: Hetong decoction. Data were expressed as mean±standard deviation (n = 10). aP < 0.001 vs Control. cP < 0.05, dP < 0.01 and bP < 0.001 vs Model.
Figure 2 Effect of HTT on serum vasopressin (AVP) and cyclic adenosine monophosphate (cAMP) concentration and intestinal aquaporin (AQP)3 and AQP8 expression in rats with loperamide-induced constipation A1-A3, B1-B3: the concentration of AVP and cAMP in serum of rats was measured using enzyme-linked immunosorbent assay (ELISA) kits. C: the protein expression of AQP3 and AQP8 in proximal and distal colon tissues of rats was determined using western blot assy. Control group was administered intragastrically with equal amount of 0.9% sodium. Model group was administered with normal saline. HTT-low (L), HTT-medium (M), HTT-high (H) groups were intragastrically given 95g/kg HTT (HTT-L), 190 g/kg HTT (HTT-M) or 380 g/kg HTT (HTT-H) twice a day for consecutive 30 d. Positive control group was given 1.60 mg/kg mosapride (MedChemExpress) lavage once every two weeks for 4 weeks. HTT: Hetong decoction. Data were expressed as mean ± standard deviation (n = 10). aP < 0.001 vs Control; bP < 0.001, cP < 0.05 and dP < 0.01 vs Model.
Figure 2 Effect of HTT on serum vasopressin (AVP) and cyclic adenosine monophosphate (cAMP) concentration and intestinal aquaporin (AQP)3 and AQP8 expression in rats with loperamide-induced constipation A1-A3, B1-B3: the concentration of AVP and cAMP in serum of rats was measured using enzyme-linked immunosorbent assay (ELISA) kits. C: the protein expression of AQP3 and AQP8 in proximal and distal colon tissues of rats was determined using western blot assy. Control group was administered intragastrically with equal amount of 0.9% sodium. Model group was administered with normal saline. HTT-low (L), HTT-medium (M), HTT-high (H) groups were intragastrically given 95g/kg HTT (HTT-L), 190 g/kg HTT (HTT-M) or 380 g/kg HTT (HTT-H) twice a day for consecutive 30 d. Positive control group was given 1.60 mg/kg mosapride (MedChemExpress) lavage once every two weeks for 4 weeks. HTT: Hetong decoction. Data were expressed as mean ± standard deviation (n = 10). aP < 0.001 vs Control; bP < 0.001, cP < 0.05 and dP < 0.01 vs Model.
Figure 3 The effect of HTT on proximal colon aquaporin (AQP)3 and AQP8 expression in rats with loperamide-induced constipation A1, B1: control group; A2, B2: model group; A3, B3: HTT-L; A4, B4: HTT-M; A5, B5: HTT-H; A6, B6: positive control. Representative images of IHC analysis for AQP3 and AQP8 expression in proximal intestines (× 200, n = 10). Brown or dark brown represents AQP3/8 positive, and blue represents negative. The darker the brown color, the higher the expression level of AQP3/8. Control group was administered intragastrically with equal amount of 0.9% sodium. Model group was administered with normal saline. HTT-low (L), HTT-medium (M), HTT-high (H) groups were intragastrically given 95 g/kg HTT (HTT-L), 190 g/kg HTT (HTT-M) or 380 g/kg HTT (HTT-H) twice a day for consecutive 30 d. Positive control group was given 1.60 mg/kg mosapride (MedChemExpress) lavage once every two weeks for 4 weeks. HTT: Hetong Decoction.
Figure 3 The effect of HTT on proximal colon aquaporin (AQP)3 and AQP8 expression in rats with loperamide-induced constipation A1, B1: control group; A2, B2: model group; A3, B3: HTT-L; A4, B4: HTT-M; A5, B5: HTT-H; A6, B6: positive control. Representative images of IHC analysis for AQP3 and AQP8 expression in proximal intestines (× 200, n = 10). Brown or dark brown represents AQP3/8 positive, and blue represents negative. The darker the brown color, the higher the expression level of AQP3/8. Control group was administered intragastrically with equal amount of 0.9% sodium. Model group was administered with normal saline. HTT-low (L), HTT-medium (M), HTT-high (H) groups were intragastrically given 95 g/kg HTT (HTT-L), 190 g/kg HTT (HTT-M) or 380 g/kg HTT (HTT-H) twice a day for consecutive 30 d. Positive control group was given 1.60 mg/kg mosapride (MedChemExpress) lavage once every two weeks for 4 weeks. HTT: Hetong Decoction.
Figure 4 Effect of HTT on aquaporin (AQP)3 and AQP8 expression in NCM460 cells A, B1, B2: AQP3/8 was silenced or overexpressed in NCM460 cells using corresponding transfection vectors, then the mRNA level of AQP3/8 was measured using quantitative real-time PCR (RT-qPCR) assay. Control group was administered intragastrically with equal amount of 0.9% sodium. Model group was administered with normal saline. HTT-low (L), HTT-medium (M), HTT-high (H) groups were intragastrically given 95 g/kg HTT (HTT-L), 190 g/kg HTT (HTT-M) or 380 g/kg HTT (HTT-H) twice a day for consecutive 30 d. Positive control group was given 1.60 mg/kg mosapride (MedChemExpress) lavage once every two weeks for 4 weeks. HTT: Hetong decoction. Data are expressed as mean±standard deviation (n = 3).aP < 0.05, cP < 0.01 and eP < 0.001 vs siRNA-NC. fP < 0.001 vs Ov-NC. C-F, NCM460 cells with AQP3/8 overexpression or knockdown were exposed to the serum of rats in the control, HTT-L or HTT-H groups for 24 h before detection of AQP3/8 expression through western blot analysis. Data are expressed as mean±standard deviation (n = 3). fP < 0.001 vs Control. bP < 0.05 and f P < 0.001 vs siRNA/Ov-AQP3/8.
Figure 4 Effect of HTT on aquaporin (AQP)3 and AQP8 expression in NCM460 cells A, B1, B2: AQP3/8 was silenced or overexpressed in NCM460 cells using corresponding transfection vectors, then the mRNA level of AQP3/8 was measured using quantitative real-time PCR (RT-qPCR) assay. Control group was administered intragastrically with equal amount of 0.9% sodium. Model group was administered with normal saline. HTT-low (L), HTT-medium (M), HTT-high (H) groups were intragastrically given 95 g/kg HTT (HTT-L), 190 g/kg HTT (HTT-M) or 380 g/kg HTT (HTT-H) twice a day for consecutive 30 d. Positive control group was given 1.60 mg/kg mosapride (MedChemExpress) lavage once every two weeks for 4 weeks. HTT: Hetong decoction. Data are expressed as mean±standard deviation (n = 3).aP < 0.05, cP < 0.01 and eP < 0.001 vs siRNA-NC. fP < 0.001 vs Ov-NC. C-F, NCM460 cells with AQP3/8 overexpression or knockdown were exposed to the serum of rats in the control, HTT-L or HTT-H groups for 24 h before detection of AQP3/8 expression through western blot analysis. Data are expressed as mean±standard deviation (n = 3). fP < 0.001 vs Control. bP < 0.05 and f P < 0.001 vs siRNA/Ov-AQP3/8.
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