Da-Cheng-Qi-Decoction (DCQD) has been used in the treatment of acute pancreatitis (AP) in China for many years. The aim of the current study was to examine the principal ingredient rhubarb of DCQD and its potential link to the pancreatic repair effects in rats with AP. The pancreatitis was induced in SD rats by intraperitoneal injections of cerulein. The results showed that rhubarb significantly increased blood perfusion of pancreatic tissue, reversed mitochondrial damage, and promoted pancreatic acinar and stellate cell proliferation. In addition, the rhein (from rhubarb) had high distribution in pancreas tissue and protected mitochondria in AR42J cells via the activation of PI3K/AKT/mTOR signaling pathway and activity inhibition of AMPK (P < 0.05). The results provide some preclinical evidence on the protective effects of DCQD for the treatment of acute pancreatitis. Rhein is regarded to be the active compound of rhubarb and can be expected to be a new compound for the treatment of AP.
Severe acute pancreatitis (SAP) is a very serious systemic disease without any effective treatments. In patients with pancreatic necrosis, about 1/3~1/2 patients will present with diabetes mellitus or transit to chronic pancreatitis after SAP resolves [
DCQD is composed of rhubarb (Rheum officinale Baill.), Houpu (Magnolia officinalis Rehd), Zhishi (Fructus Aurantii Immaturus), and Mangxiao (Natrii Sulfas). Rhubarb is the principal ingredient in DCQD, exerting the major therapeutic activity of the decoction. It has been established that a systemic inflammatory response and disturbance of the pancreatic microcirculation occur at almost the same time in SAP and the process of regeneration [
Pure emodin, aloe-emodin, rhein, and chrysophanol, all of which are present in stewed rhubarb, as well as >99% pure zaltoprofen as an internal standard, were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC-grade methanol was obtained from Tedia (Ohio, USA). Acetic acid and ethyl acetate were purchased from Chongqing Chemistry Co. Ltd. (Chongqing, China). Analytical-grade ammonium acetate, sodium hydroxide, and hydrochloric acid were purchased from Chengdu Kelong Chemical Reagent Factory (Chengdu, China). All other reagents were obtained from Sigma (New Jersey, USA).
Rhubarb (stewed) granules were purchased from Chengdu Green Herbal Pharmaceutical Co. Ltd. (Chengdu, China). Extraction yield was approximately 5% (w/w, dried extract/crude herb). Before being orally administered to rats, the stewed rhubarb granules were reconstituted with water to a concentration of 1g/ml. The specimens used in this study were stored in Voucher specimens of rhubarb number 2013-0104 and deposited in the Laboratory of Pharmacology, Sichuan University.
Rhubarb granules were reconstituted in distilled water to a concentration of 6 mg/ml. The mixture was centrifuged at 3000 rpm for 10 min, and 10
Male Sprague-Dawley (SD) rats (220 ± 50 g) were maintained under controlled environmental conditions and fasted for 24h with free access to water prior to experiments. All experiments were approved by the Animal Ethics Committee of Sichuan University and carried out in accordance with university guidelines. Pancreatitis was induced in SD rats by 7 intraperitoneal (IP) injections at 1h intervals of 50
On the 1st, 2nd, 5th, and 8th days following the injection of saline or cerulein, the animals were anesthetized, and the abdomen was opened. Pancreases were exposed for the measurement of pancreatic blood flow using a laser Doppler flowmeter (PF5010, Sweden). Pancreatic blood flow was measured in five different portions of the pancreas and the area of laser emission of the probe was about 1mm2, while the depth of measurement reached about 3mm. These were recorded and presented as the mean perfusion (PU) from five different points (PU).
Plasma amylase levels were determined at 37°C by means of an enzymatic assay (Jiancheng, Nangjing, China) with a spectrophotometer. All plasma samples were assayed in duplicate, and the results were averaged at the end of each experiment.
Regeneration of the damaged pancreas was monitored histologically for the reappearance of zymogen granule-containing acinar cells and the organization of these cells into acini. Replication of pancreas cells was determined by immunohistologic detection of cells expressing Ki-67, as a marker of proliferation, transforming growth factor beta 1 (TGF-
Small specimens (about 1mm3) of pancreatic tissue (three from each animal) were immediately fixed in 3.6% glutaraldehyde in 0.1mol/L cacodylate buffer (pH7.4) for 3h and, after washing in the buffer, post fixed in 2% osmium tetroxide for 1h. The samples were dehydrated in alcohol and propylene oxide and then embedded in Epon812. The ultrathin sections were cut from each block on a Reichert ultramicrotome, stained with lead citrate and uranyl acetate, and studied under an Opton900 PC transmission electron microscope field by field. Fifty to sixty electron micrographs of the most characteristic changes from each group were made. The determination of pathology was made blind.
The rat pancreatic acinar AR42J cells (ATCC, Rockville, MD, USA) were cultured in F12K medium containing 20% FBS, 100U/mL penicillin, and 100U/mL streptomycin under standard conditions (37°C and 5% CO2). AR42J cells (1million/mL) seeded in flat-bottom 6-well plates were divided into the following groups: (1) control group, in which cells were cultured in medium alone; (2) AP model group, in which cells were cultured in medium containing cerulein (10−8M, Sigma); (3) treatment group, in which cells were cultured in medium containing cerulein and rhein (1uM), respectively. The concentration of rhein in cell culture is determined by our previous study on plasma and tissue concentrations result of rhein in rats after administration of DCQD [
Coincubated with rhein and cerulein for a further 16h, the cells were collected by centrifugation, the supernatant was removed, and the fixed solution was added slowly to fix the 3% glutaraldehyde to maintain the mass of cells. After dehydration and embedding, cells were stained with uranyl acetate and lead citrate and then examined using an electron microscope.
The effect of rhein on the mitochondrial membrane potential of pancreatic AR42J cells was measured using mitochondrial membrane potential fluorometric assay kits. MMP is a marker of mitochondrial oxidative phosphorylation activity that can be assessed using fluorescent probes accumulating in mitochondria depending on the MMP. For detection of MMP in this study, the fluorescent dye TMRM (tetramethylrhodamine methyl ether, 1uM), which is concentrated by respiring mitochondria, was employed.
AR42J cells were resuspended in 1ml complete PBS followed by liquid nitrogen refrigeration. Thawing (6-7 times) until the cells dissolved at 37°C, centrifuged at 3000×g for 10min, the supernatant was collected for cryopreservation. AMPK (Adenosine 5‘-monophosphate activated protein kinase), PI3K (phosphatidylinositol 3-kinase), and AKT (protein kinase B) optical density were measured by ELISA kits (Bassett Weiss Biotech, Shenzhen, China) complying with the manufacturer’s protocols on enzyme labeling instrument. The OD value of each sample was determined according to the standard curve and the corresponding curve equation to calculate the concentration of each index in the sample.
Differences in results obtained with stewed rhubarb and rhein treatments were tested for statistical significance using one-way ANOVA. Differences with an associated P value of 0.05 or less were considered significant.
Major components of stewed rhubarb include the four anthraquinones with known compounds present in the following relative abundance: emodin > aloe-emodin > rhein > chrysophanol (Figure
Chromatogram of rhubarb sample emodin, aloe-emodin, rhein, chrysophanol, and internal standards (1,8-dioxyanthraquinone).
In AP, inflammation of the pancreatic cells leads to elevated levels of plasma amylase. Plasma amylase was significantly higher in the AP model than that in rats of the sham group. In rats given rhubarb, amylase in serum was lower than that observed in AP rats (P < 0.05) (Table
Levels of plasma amylase in the three groups (IU/L, m ± SD) (n = 6).
Sham | AP | Treatment | |
---|---|---|---|
AMY (IU/L) | 992 ± 221 | 2077 ± 585 | 1458 ± 450 |
The pancreatic tissue perfusion was significantly reduced after the first two days in the AP group. Rhubarb significantly increased pancreatic tissue blood perfusion at day 2 (510 ± 187 versus 230 ± 68). Between day 5 and day 8, pancreatic tissue blood perfusion showed no difference among the normal, AP, and treatment groups (Figure
Effect of rhubarb on the pancreatic blood perfusion on different days in three groups. C: control group; AP: acute pancreatitis group; T: rhubarb treated group.
Using electron microscopy, we found obvious morphological changes in the model group: more lysosomes with more swallowed organelles after necrosis, rough endoplasmic reticulum mild expansion or disappearance, severe mitochondrial damage where ridges ruptured or disappeared, and mitochondrial swelling on the 2nd day. The rhubarb groups showed a significant reversal of morphology: the intracellular vacuoles decreased or disappeared with a small amount of neutral cells and eosinophils, especially the reversal of mitochondrial morphology from the AP group. On the 5th day, swelling of mitochondria with a large number of lymphocytes was still found in rhubarb groups. However, the overall shape of the acinar ultrastructure showed no difference among all the groups on the 8th day (Figure
Acinar cell regeneration and ultrastructure were observed from the electron microscopy. (a) In AP rat, there were more lysosomes with more swallowed organelles after pancreas necrosis, rough endoplasmic reticulum mild expansion or disappearance, severe mitochondrial damage where ridges ruptured or disappeared, and mitochondrial swelling (arrows) on the 2nd day. (b) Ultrastructure of acinar cells from the electron microscopy was not detected with obvious morphological changes in the rhubarb treated group on the 2nd day. (c) On Day 5, swelling of mitochondria with a large number of lymphocytes is still found in rhubarb groups. (d) The overall shape of the acinar ultrastructure was ameliorated on the 8th day in the rhubarb treated group. Original magnification: ×1000 (a-d).
The positive actin monoclonal antibody staining reflects stellate cells in the matrix, while Ki-67 staining reflects the pancreatic acinar cell nuclei proliferation. TGF-
Immunohistochemical analysis of pancreatic tissues of rats for the expression of actin, Ki-67, and transforming growth factor beta1 (TGF-
In the model group, in which AR42J cells were cultured in medium containing cerulein (10−8 M), more necrosis organelles were contained in the lysosomes after being swallowed. Rough endoplasmic reticulum was dilated or disappeared. Mitochondria swelled and ridges were broken or disappeared, showing vacuolization. After 16h of treatment of rhein, mitochondrial swelling and spinal fracture were reduced. Rhein can significantly improve cell ultrastructure, especially the improvement of mitochondrial morphology (Figure
Acinar cell regeneration and ultrastructure were observed from the electron microscopy. (a) AR42J cells: the structures of organelles seemed almost intact. (b) Model group, in which AR42J cells were cultured in medium containing cerulein (10−8 M): mitochondria were damaged where ridges ruptured or disappeared (arrows). (c) After 16h of treatment of rhein, mitochondrial swelling and fracture were reduced.
The fluorescence ELISA data showed that, compared with the control group, cerulein stimulation decreased the mitochondrial membrane potential of AR42J cells, indicating that cerulein damaged the structure and function of mitochondria. After treatment with rhein for 16 hours, the potential of the treated group was obviously restored compared with the model group (P < 0.05), indicating that rhein can effectively lighten the cerulein that caused mitochondrial damage and improve mitochondrial function (Figure
Effect of rhein on the mitochondrial membrane potential of AR42J cells in three groups. C: control group; AP: acute pancreatitis group; T: rhein treated group.
Expression of AMPK, PI3K, AKT, and mTOR (mammalian target of rapamycin) was detected using ELISA. As a cellular energy sensor responding to low ATP levels, AMPK activation negatively regulates ATP-consuming biosynthetic processes such as protein synthesis to improve the energy metabolism when pancreatitis happened. The PI3K/AKT/mTOR signaling pathway exerts a significant effect on the regulation of metabolism, cell growth, and survival [
Expression of AMPK, PI3K, AKT, and mTOR was detected using ELISA in three groups. C: control group; AP: acute pancreatitis group; T: rhein treated group.
The clinical sequence and limited pathologic data suggest that the symptoms of mild and severe pancreatitis are comparable in animal models and humans. Both mild and severe forms of the disease have a similar progression and outcome, although its progress is usually more rapid in rodents than in humans [
AP is initiated by intracellular activation of pancreatic proenzymes and autodigestion of the pancreas. Destruction of the pancreatic parenchyma first induces a local inflammatory reaction [
We also observed that induction of AP by cerulein caused an initial reduction of pancreatic blood flow on the 2nd day followed by a subsequent increase in this parameter. Rhubarb caused a reversal of the cerulein-induced fall in pancreatic blood flow in the early stages of the regeneration process. The mechanism of this circulatory effect is likely associated with the well-known anti-inflammatory, blood circulation promotion, and blood stasis removal effects of rhubarb [
Other experimental studies have shown that administration of growth factors attenuates pancreatic damage and accelerates pancreatic recovery [
Consistent with our results, a study conducted by Halbrook et al. also showed that short-term blockage of AMPK signaling restored exocrine tissue and dramatically reduced fibrosis in established pancreatitis [
In conclusion, we did in vivo studies of rhubarb and in vitro experiments on one major phytochemical component of rhein from rhubarb to test their effects on pancreatic regeneration. Taking the two sets of results together, rhubarb improved the severity of pancreatitis in cerulein-induced experimental AP in rats and promoted the spontaneous repair and regeneration processes of pancreatic tissue. In addition, the anthraquinone, rhein, had higher penetration into the pancreas in the AP disease state (our previous research), which contributes to the ability of rhubarb to promote regeneration of the pancreas through mitochondrial protection. Our findings are evidence that rhein exerts a pancreatic protective effect via the activation of the PI3K/AKT/mTOR signaling pathway and activates inhibition of AMPK in AR42J cells suffering from cerulein; rhein is therefore a potential new compound for the treatment of AP.
It needs to be stressed that Chinese herbs have many pharmacological substances and therefore have multiple therapeutic effects. Another question is which compound(s) of rhubarb is (are) the most active? A major limitation of the present study is that we conveniently chose the most abundant substance in pancreatic tissue (from rhubarb) to test, but the screening for other possible active compound(s) responsible for regeneration of pancreatic acinar cells was not established in AR42J cells. Future studies utilizing pancreatic AR42J cells to determine the active compound(s) of rhubarb and its mechanism of action may provide more complete answers, and these experiments are being planned.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare no conflicts of interest.
Dr. Jianlei Zhao, Zhu Yuan, and Mei Tang supervised the design of this study and Jianlei Zhao wrote the first draft of the paper. The authors thank Dr. Jin Xiang who contributed to the technical support with HPLC. Dr. George Li. contributed to and approved the final manuscript. Litao Liu and Wenbi Xiong assume responsibility for the animal experiments and the accuracy of the data analysis. All authors contributed to and approved the final manuscript.
This research was supported by the National Natural Science Foundation of China (grant: 81370091).