Previous study demonstrated that Chang Run Tong (CRT) could partly restore the colon remodeling in streptozotocin- (STZ-) induced diabetic rats. Here we investigated the mechanisms of such effects of CRT. Diabetes was induced by a single injection of 40 mg/kg of STZ. CRT was poured into the stomach by gastric lavage once daily for 60 days. The remodeling parameters were obtained from diabetic (DM), CRT treated diabetic (T1, 50 g/kg; T2, 25 g/kg), and normal (Con) rats. Expressions of advanced glycation end product (AGE), AGE receptor, transforming growth factor-
Constipation is very common in the general population [
The traditional Chinese medicine has good clinical effects on constipation [
The gastrointestinal (GI) tract is functionally subjected to dimensional changes. Hence, biomechanical properties such as the stress-strain relationship are of particular importance [
Advanced glycation end products (AGEs) are formed physiologically, increased with aging, and accelerated in diabetes [
In order to investigate the mechanism of CRT in the improvement on the morphometric and biomechanical remodeling of the colon in streptozotocin- (STZ-) induced diabetic rats, the expressions of advanced glycation end product (AGE), AGE receptor (RAGE), transforming growth factor-
Forty male Sprague-Dawley rats weighing 220–250 g were included in this study. Thirty rats were made diabetic by a single tail-vein injection of 40 mg/kg STZ (Sigma-Aldrich, China). This dose of STZ resulted in a random blood glucose level (≥16.7 mmol/L) in 90% of the rats 7 d after injection. The remaining 10% of rats were excluded from this study. Twenty-seven STZ-induced diabetic rats were subdivided into three groups (
CRT is composed of
The body weight and blood glucose levels were measured at 2-week intervals after the start of the experiment. The experimental period was 60 d. At the ending of the experiment, the rats fasted overnight and then were anesthetized with 4% chloral hydrate (10 mL/kg, ip). Following laparotomy, the whole colon was harvested. After the lumen of the segments was gently cleaned with saline, the length and the wet weight were measured. The colonic segment was divided into three parts: A 2 cm long tissue was cut from proximal end of the segments and fixed in 10% formalin for immunohistochemistry examination. Then a 1 cm long part was cut and used for the zero-stress state experiment and the remaining part was used for the distension test. The results of zero-stress state and the distension test have been reported in our previous paper [
The tissue samples for immunohistochemistry were fixed in 10% phosphate-buffered formalin for 24 h and embedded in paraffin. Five-micron sections were cut perpendicular to the mucosa surface and placed in a water bath at 40°C. Thereafter, sections were transferred onto pretreated microscopic slides which electrostatically attracted formalin fixed tissue and bound them to the slides. After drying the slides completely at room temperature, they were treated in an oven at 37°C overnight to enhance the attachment of tissue to the slides. The sections were deparaffinized two times in xylene, 15 min per time, and rehydrated in 100%, 95%, 90%, 80%, 70%, 60%, and 50% ethanol two times, respectively, 3 sec per time, followed by rinsing for 10 min and washing in 0.01 M PBS (pH 7.4).
After treatment with H2O2 (3% in ethanol, room temperature, 15 min) and proteinase K (100
The primary anti-RAGE antibody was produced in rabbits immunized with a synthetic peptide corresponding to a sequence at the N-terminal of human RAGE (Sigma). Only two amino acids are different from the related rat sequence. Instead of treating sections with proteinase K for RAGE immunostaining, the sections were boiled in 10 mM citrate buffer (pH 6.0) for 18 min for retrieving antigen. Normal rat lung was used as positive control since RAGE is highly expressed in the lung [
The primary TGF-
AGE, RAGE, TGF-
The data were representative of a normal distribution and accordingly the results were expressed as means ± SEM. Student’s
The blood glucose, body weight, the wet weight per unit length, no-load wall thickness, and cross section wall area measured at the end of the experiment are shown in Table
Parameters of glucose, body weight, and morphometry of colon.
Con | DM | T1 | T2 | |
---|---|---|---|---|
Body weight (g) | 443.69 ± 5.01 | 231.22 ± 5.05 |
268.33 ± 6.23 |
237.11 ± 3.75 |
Blood glucose (mmol/L) | 5.06 ± 0.04 | 30.23 ± 0.41 |
24.04 ± 0.56 |
32.21 ± 0.33 |
Wall thickness (mm) | 0.98 ± 0.05 | 1.23 ± 0.04 |
1.13 ± 0.05 |
1.11 ± 0.03 |
Wall area (mm2) | 11.04 ± 0.81 | 16.55 ± 0.98 |
13.95 ± 0.62 |
15.51 ± 0.92 |
Wet weight per unit (g/cm) | 0.13 ± 0.02 | 0.15 ± 0.02 |
0.15 ± 0.02 |
0.16 ± 0.02 |
Compared with Con:
The biomechanical parameters of the colon segment obtained from previous study [
Parameters of biomechanical properties of colon.
Con | DM | T1 | T2 | |
---|---|---|---|---|
Opening angle (degree) | 101.45 ± 8.84 | 223.66 ± 21.93 |
159.17 ± 12.31 |
195.341 ± 17.22 |
Inner residual strain (unitless) | −0.21 ± 0.02 | −0.29 ± 0.03 |
−0.26 ± 0.02 |
−0.27 ± 0.03 |
Outer residual strain (unitless) | 0.12 ± 0.02 | 0.22 ± 0.03 |
0.12 ± 0.01 |
0.26 ± 0.03 |
Circumferential constant |
1.13 ± 0.16 | 2.12 ± 0.28 |
1.87 ± 0.3162 |
2.61 ± 0.38 |
Longitudinal constant |
25.48 ± 4.12 | 52.34 ± 8.73 |
36.13 ± 2.48 |
44.76 ± 5.59 |
Compared with Con:
The fraction of AGE, RAGE, TGF-
Fractions of AGE, RAGE, TGF-
Proteins | Layers | Con | DM | T1 | T2 |
---|---|---|---|---|---|
AGE | Mucosa | 0.135 ± 0.027 | 0.196 ± 0.028 |
0.085 ± 0.019## | 0.138 ± 0.029# |
Muscle | 0.147 ± 0.040 | 0.474 ± 0.065 |
0.173 ± 0.069## | 0.452 ± 0.099 |
|
Submucosa | 0.032 ± 0.011 | 0.167 ± 0.035 |
0.049 ± 0.012## | 0.094 ± 0.012 |
|
|
|||||
RAGE | Mucosa | 0.054 ± 0.007 | 0.113 ± 0.040 |
0.052 ± 0.017## | 0.150 ± 0.041 |
Muscle | 0.189 ± 0.061 | 0.378 ± 0.093 |
0.162 ± 0.042## | 0.423 ± 0.087 |
|
Submucosa | 0.059 ± 0.033 | 0.263 ± 0.078 |
0.071 ± 0.021## | 0.226 ± 0.047 |
|
|
|||||
TGF- |
Mucosa | 0.061 ± 0.014 | 0.119 ± 0.036 |
0.065 ± 0.012## | 0.194 ± 0.062 |
Muscle | 0.211 ± 0.028 | 0.331 ± 0.072 |
0.191 ± 0.031# | 0.344 ± 0.102 |
|
Submucosa | 0.097 ± 0.019 | 0.173 ± 0.051 |
0.099 ± 0.022## | 0.275 ± 0.083 |
|
|
|||||
TGF- |
Mucosa | 0.116 ± 0.025 | 0.232 ± 0.066 |
0.141 ± 0.024# | 0.278 ± 0.035 |
Muscle | 0.188 ± 0.008 | 0.422 ± 0.079 |
0.227 ± 0.022## | 0.403 ± 0.044 |
|
Submucosa | 0.231 ± 0.023 | 0.379 ± 0.069 |
0.222 ± 0.017# | 0.399 ± 0.021 |
Compared with Con:
The representative samples of immunohistochemical staining for AGE, RAGE, TGF-
In order to analyze the expressions of AGE, RAGE, TGF-
The relation between expressions of AGE, RAGE TGF-
Proteins | Linear regression equation |
|
|
---|---|---|---|
AGE | AGE-M = 0.0651 + (0.00311 |
0.527 | 0.010 |
AGE-M = −0.0297 + (0.0115 |
0.553 | 0.006 | |
AGE-M = 0.0690 + (0.375 |
0.576 | 0.004 | |
AGE-M = 0.0472 + (0.000549 |
0.536 | 0.008 | |
AGE-Mu = 0.0367 + (0.0134 |
0.673 | <0.001 | |
AGE-Mu = −0.540 + (0.802 |
0.479 | 0.021 | |
AGE-Mu = −0.304 + (0.0450 |
0.640 | 0.001 | |
AGE-Mu = 0.0765 + (1.494 |
0.679 | <0.001 | |
AGE-Mu = −0.0104 + (0.00219 |
0.632 | 0.001 | |
AGE-Mu = 0.167 + (0.0910 |
0.430 | 0.040 | |
AGE-Sub = 0.00190 + (0.00426 |
0.636 | 0.001 | |
AGE-Sub = 0.223 − (0.000435 |
0.498 | 0.015 | |
AGE-Sub = 0.00176 + (168.500 |
0.462 | 0.026 | |
AGE-Sub = −0.267 + (0.332 |
0.590 | 0.003 | |
AGE-Sub = −0.121 + (0.0153 |
0.648 | <0.001 | |
AGE-Sub = 0.00494 + (0.526 |
0.711 | <0.001 | |
AGE-Sub = −0.0129 + (0.000694 |
0.596 | 0.003 | |
AGE-Sub = 0.0254 + (0.0376 |
0.529 | 0.009 | |
|
|||
RAGE | RAGE-M = 0.0114 + (0.00418 |
0.544 | 0.007 |
RAGE-M = 0.229 − (0.000415 |
0.435 | 0.038 | |
RAGE-M = −0.00701 + (201.874 |
0.507 | 0.014 | |
RAGE-M = −0.231 + (0.310 |
0.504 | 0.014 | |
RAGE-M = −0.111 + (0.0153 |
0.636 | 0.001 | |
RAGE-M = 0.0507 + (0.340 |
0.421 | 0.046 | |
RAGE-M = 0.00428 + (0.000625 |
0.529 | 0.009 | |
RAGE-Mu = 0.0833 + (0.0107 |
0.565 | 0.005 | |
RAGE-Mu = 0.631 − (0.00104 |
0.443 | 0.034 | |
RAGE-Mu = −0.0898 + (0.0296 |
0.501 | 0.015 | |
RAGE-Mu = 0.0857 + (0.00148 |
0.510 | 0.013 | |
RAGE-Mu = 0.172 + (0.0826 |
0.438 | 0.037 | |
RAGE-Mu = 0.184 + (0.00456 |
0.416 | 0.048 | |
RAGE-Sub = −0.0181 + (0.00827 |
0.630 | 0.001 | |
RAGE-Sub = 0.431 − (0.000889 |
0.546 | 0.007 | |
RAGE-Sub = −0.0568 + (403.042 |
0.593 | 0.003 | |
RAGE-Sub = −0.547 + (0.576 |
0.549 | 0.007 | |
RAGE-Sub = −0.194 + (0.0258 |
0.627 | 0.001 | |
RAGE-Sub = −0.0213 + (0.00117 |
0.582 | 0.004 | |
RAGE-Sub = 0.0663 + (0.00336 |
0.441 | 0.035 | |
|
|||
TGF- |
TGF- |
0.564 | 0.009 |
TGF- |
0.431 | 0.040 | |
TGF- |
0.474 | 0.022 | |
TGF- |
0.447 | 0.049 | |
|
|||
TGF- |
TGFR-M = 0.0894 + (0.00425 |
0.443 | 0.034 |
TGFR-M = 0.330 − (0.000495 |
0.435 | 0.038 | |
TGFR-M = 0.0294 + (275.036 |
0.531 | 0.009 | |
TGFR-M = −0.197 + (0.346 |
0.447 | 0.033 | |
TGFR-M = −0.114 + (0.0208 |
0.609 | 0.002 | |
TGFR-M = 0.0637 + (0.684 |
0.654 | <0.001 | |
TGFR-M = 0.0687 + (0.000705 |
0.431 | 0.040 | |
TGFR-Mu = 0.123 + (0.00785 |
0.627 | 0.001 | |
TGFR-Mu = 0.522 − (0.000753 |
0.507 | 0.013 | |
TGFR-Mu = 0.113 + (334.580 |
0.495 | 0.016 | |
TGFR-Mu = −0.0774 + (0.0264 |
0.593 | 0.003 | |
TGFR-Mu = 0.188 + (0.645 |
0.473 | 0.023 | |
TGFR-Mu = 0.104 + (0.00119 |
0.559 | 0.006 | |
TGFR-Sub = 0.190 + (0.00488 |
0.479 | 0.021 | |
TGFR-Sub = 0.449 − (0.000506 |
0.419 | 0.047 | |
TGFR-Sub = 0.160 + (248.342 |
0.451 | 0.031 | |
TGFR-Sub = 0.0479 + (0.0176 |
0.486 | 0.019 | |
TGFR-Sub = 0.208 + (0.526 |
0.473 | 0.023 | |
TGFR-Sub = 0.185 + (0.000700 |
0.415 | 0.049 |
Notes: M, mucosa; Mu, muscle; Sub, submucosa; Glu, glucose; BW, body weight; Wt, weight per unit length; W-h, wall thickness; W-a, wall area; Res-in, inner residual strain; Res-out, outer residual strain; OA, opening angle, C-
In order to determine the main contributor of AGE, RAGE, TGF-
Table
Multiple linear regression analysis for the relation between expressions of AGE, RAGE, TGF-
Parameter | Multiple linear regression equation |
|
|
|
Independent |
|
---|---|---|---|---|---|---|
Glucose | Glu = 8.960 + (57.741 |
0.696 | 3.201 | 0.032 | AGE-M |
0.103 |
Glu = 12.108 + (26.395 |
0.762 | 4.693 | 0.007 | AGE-Mu |
0.037 | |
Glu = 13.082 + (48.264 |
0.748 | 4.314 | 0.010 | AGE-Sub |
0.197 | |
|
||||||
Wall area | W-a = 10.385 + (12.595 |
0.797 | 5.909 | 0.002 | AGE-M |
0.160 |
W-a = 10.615 + (10.314 |
0.706 | 3.377 | 0.027 | AGE-Mu |
0.016 | |
W-a = 10.180 + (21.961 |
0.746 | 4.258 | 0.011 | AGE-Sub |
0.062 | |
|
||||||
Outer residual strain | res-out = 0.0647 + (0.557 |
0.808 | 6.411 | 0.002 | AGE-M |
0.039 |
res-out = 0.104 + (0.377 |
0.697 | 3.220 | 0.032 | AGE-Mu |
0.005 | |
res-out = 0.0843 + (1.013 |
0.738 | 4.076 | 0.013 | AGE-Sub |
0.007 | |
|
||||||
Opening angle | OA = 100.128 + (219.169 |
0.643 | 2.394 | 0.081 | AGE-M |
0.337 |
OA = 81.341 + (200.133 |
0.814 | 6.690 | 0.001 | AGE-Mu |
0.007 | |
OA = 66.919 + (310.291 |
0.716 | 3.576 | 0.022 | AGE-Sub |
0.204 | |
|
||||||
C-constant |
C- |
0.499 | 1.127 | 0.384 | AGE-M |
0.587 |
C- |
0.608 | 1.991 | 0.132 | AGE-Mu |
0.145 | |
C- |
0.607 | 1.985 | 0.133 | AGE-Sub |
0.119 | |
|
||||||
L-constant |
L- |
0.560 | 1.555 | 0.226 | AGE-M |
0.028 |
L- |
0.628 | 2.119 | 0.113 | AGE-Mu |
0.176 | |
L- |
0.526 | 1.301 | 0.309 | AGE-Sub |
0.949 |
Notes: M, mucosa; Mu, muscle; Sub, submucosa; Glu, glucose; W-a, wall area; Res-out, outer residual strain; OA, opening angle, C-
Interrelation among AGE, RAGE, TGF-
Interrelation among AGE, RAGE, TGF-
Parameter | Multiple linear regression equation |
|
|
|
Independent |
|
---|---|---|---|---|---|---|
Mucosa-AGE | AGE-M = 0.0812 + (0.372 |
0.431 | 1.026 | 0.421 | RAGE-M |
0.173 |
|
||||||
Muscle-AGE | AGE-Mu = −0.0919 + (0.640 |
0.726 | 5.004 | 0.007 | RAGE-Mu |
0.012 |
|
||||||
Submucosa-AGE | AGE-Sub = −0.0559 + (0.294 |
0.707 | 4.492 | 0.011 | RAGE-Sub |
0.014 |
|
||||||
Mucosa-RAGE | RAGE-M = 0.0390 + (0.271 |
0.785 | 7.235 | 0.001 | AGE-M |
0.173 |
|
||||||
Muscle-RAGE | RAGE-Mu = 0.232 + (0.477 |
0.752 | 5.846 | 0.003 | AGE-Mu |
0.012 |
|
||||||
Submucosa-RAGE | RAGE-Sub = 0.302 + (0.985 |
0.720 | 4.842 | 0.008 | AGE-Sub |
0.014 |
|
||||||
Mucosa-TGF- |
TGF-M = 0.0934 + (0.166 |
0.501 | 1.511 | 0.241 | AGE-M |
0.660 |
|
||||||
Muscle-TGF- |
TGF-Mu = 0.145 + (0.110 |
0.664 | 3.553 | 0.026 | AGE-Mu |
0.576 |
|
||||||
Submucosa-TGF- |
TGF-Sub = 0.212 + (0.384 |
0.670 | 3.669 | 0.0242 | AGE-Sub |
0.336 |
|
||||||
Mucosa-TGF- |
TGFR-M = 0.0521 − (0.0329 |
0.778 | 6.882 | 0.002 | AGE-M |
0.903 |
|
||||||
Muscle TGF- |
TGFR-Mu = 0.145 + (0.129 |
0.785 | 7.245 | 0.001 | AGE-Mu |
0.329 |
|
||||||
Submucosa-TGF- |
TGFR-Sub = 0.366 + (0.361 |
0.757 | 6.055 | 0.003 | AGE-Sub |
0.272 |
Notes: M, mucosa; Mu, muscle; Sub, submucosa.
Previously we demonstrated that CRT could restore the morphometric and biomechanical remodeling of colon in streptozotocin- (STZ-) induced diabetic rats [
Diabetic GI complications are common in long-standing diabetes [
AGEs and RAGE accumulated during the development of DM are associated with cardiovascular complication [
Transforming growth factor- (TGF-)
Analysis for interrelation among AGE, RAGE, TGF-
We have previously demonstrated that the histomorphological and biomechanical remodeling occurred in the diabetic rat model [
Data is lacking in relation to the association between TGF-
Clinically we found that CRT decoction was very effective to treat senile constipation. Studies have shown that
Furthermore, we demonstrated that high dose of CRT could significantly decrease the blood glucose level in the diabetic rats [
STZ-induced diabetes upregulated the expression of AGE, RAGE, TGF-
No potential conflict of interests relevant to this paper was reported.
Hong Sha, Dong Zhao, and Jingbo Zhao designed and performed experiments, analyzed data, and wrote the paper. Xiaolin Tong and Hans Gregersen contributed to the discussion and reviewed the paper. Jingbo Zhao and Xiaolin Tong supervised the study. Hans Gregersen contributed to the language editing. Jingbo Zhao had full access to all the data in the study and takes responsibility for the integrity of data and the accuracy of data analysis.
The study was financially supported by National Key Basic Research Program of China (973), no. 2010CB530600, and Karen Elise Jensen foundation (Project no. 903959).