Huqi San-Evoked Rat Colonic Anion Secretion through Increasing CFTR Expression

Huqi San (HQS) is a Chinese herbal preparation of eight medicinal herbs that promote diuresis, detoxification, blood circulation, and cholestasis. Defects in transporter expression and function can cause cholestasis and jaundice. However, the mechanism of the cholestasis underlying HQS effects, especially on the gastrointestinal tract ion secretion, has not been elucidated. Real-time RT-PCR and Western blotting were used to study the expression and localization of cystic fibrosis transmembrane conductance regulator (CFTR) and α-ENaC in rat alimentary tract, and then the effect of HQS on the ion transport in rat distal colon mucosa was investigated using the short-circuit current (I SC) technique. The results showed that pretreatment with HQS significantly enhanced mRNA transcripts and protein content of CFTR in liver and distal colon but not α-ENaC in alimentary organs. HQS increases I SC and decreases the transepithelial resistance. Pretreatment with epithelial Na+ channel blocker did not affect the I SC responses elicited by HQS, but removal of extracellular Cl− or pretreatment with Cl− channel or Na+-K+-2Cl− cotransporter blocker inhibited HQS-elicited I SC responses. These findings demonstrated that HQS, RA, and RP can stimulate Cl− secretion in the distal colon by increasing the mRNA transcripts and protein content of CFTR in liver and distal colon.


Background
Huqi San (HQS) is a Chinese herbal preparation of eight medicinal herbs, including Ramulus Visci, Radix Astragali seu Hedysari, Radix Curcumae, Radix Salviae Miltiorrhizae, Spica Prunellae, Semen Persicae, Semen Cuscutae, and Radix Sophorae Flavescentis, purchased from Tongrentang (Beijing, China) and authenticated by Professor Wen Wang, a botanist at Xuanwu Hospital, Beijing, China. HQS supplements qi and tonifies the kidney. HQS, especially its principal drug Ramulus Visci alkali (RA), the major active constituent of mistletoe extracts, is widely used for the treatment of tumour [1], hypertensive rats, and renal hypertensive dogs [2]. Previous studies showed that RA can inhibit Ca 2+ mobilization from intracellular stores [3], block and reverse hepatocarcinogenesis [1], gynecological and breast cancer treatment [4], and even enhance immunosurveillance to prevent intestinal infections or other intestinal pathologies by the induction of cytokines in intestinal epithelial cells [5]. As an adjuvant, polysaccharides of Ramulus Visci (RP) can inhibit cancer cell proliferation and promote cancer cell apoptosis in vivo [6]. Our previous experiment has confirmed the effect of HQS on hepatocarcinogenesis [7]. Laboratory tests revealed hepatic cell damage with cholestasis, lipid abnormalities, and hypocholesterolemia with cystic fibrosis associated with liver disease as the only manifestation of cystic fibrosis [8]. Cholangiocytes alkalinize and dilute canalicular bile through the secretion of a bicarbonate rich fluid. Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel expressed in biliary tract, is the major driving force for this ductular secretion [9,10]. Both human and animal studies have provided evidence that any impairment in the expression and/or function of these different hepatobiliary transporters may lead to cholestatic disorders [11]. The disruption and dysregulation of this excretory pathway may result in cholestasis [12] and lead to intrahepatic accumulation of bile acids and other toxic compounds with progression of hepatic pathological changes [13]. Although the transcriptional regulation of hepatic organic anion transporters by liver-enriched hepatocyte nuclear factors and ligand activated nuclear receptors is the key to understand the molecular mechanisms of cholestasis [14], the transporter changes at a transcriptional level may represent potential targets for therapy [14]. In this study we investigate the effects of HQS, RA, and RP on hepatic organic anion transporter regulation in the liver, distal colon, and pancreas of rat.

Preparation of Alkalies and
Polysaccharides. The regular experiment method was performed for qualitative analyses of the alkalies or polysaccharides in Ramulus Visci. Ramulus Visci was ground to powder. Before soaking in acidity aqueous for 48 h, alkalies in Ramulus Visci (RA) were proceeded to precipitate with the alkali and then the insoluble were filtered. The partial precipitates were achieved using different alkalinity ratios to get the total alkali, and the polysaccharides of Ramulus Visci (RP) were prepared according to the previous report [16].

Solutions and Reagents.
Hank's balanced salt solution (HBSS), glibenclamide, amiloride hydrochloride, bumetanide, and forskolin are from Sigma (St. Louis, MO, USA). The stock solutions of glibenclamide, forskolin, and bumetanide were prepared in dimethyl sulfoxide (DMSO). Final concentrations of DMSO never exceeded 0.1% (v/v). Preliminary experiments indicated that the vehicle did not change any baseline electrophysiological parameters.

Tissue Preparation.
Animal protocols followed guidelines established by the NIH and were approved by Animal Care and Use Committee, Capital Medical University. Male Sprague-Dawley rats (Laboratory Animal Services Center, Capital Medical University) ranging from 135 to 150 g (6 weeks old) had free access to standard rodent laboratory food and water until the day of the experiments. Eighty rats were equally divided (20 rats/group) into HQS therapeutic group (8 g/kg body weight), RA therapeutic group (10 mg/kg body weight), RP therapeutic group (10 mg/kg body weight), and control group with the physiological saline. All the drugs were given through intragastric administration for seven days in vivo experiment. The animals were killed by cervical dislocation. The distal colon was removed and defined as the ca. 7 cm long segment proximal to the lymph node (typically situated 3 cm apart from the anus). Then the distal colon was divided into 4 segments, which were cut along the mesenteric border into a flat sheet and flushed with ice-cold Kreb's-Henseit solution (K-HS). The tissue was pinned flat with the mucosal side down in a Sylgard-lined petri dish containing ice-cold oxygenated solution. The colon was longitudinally cut close to the mesentery, and the serosal muscle layers were carefully stripped away by blunt dissection to obtain a mucosa preparation. All the herbs and the routine drugs were supplied by means of directly apical or basolateral side in Ussing chamber system.

Short-Circuit Current Measurement.
The short-circuit current was measured in vitro in Ussing chambers. Flat sheet of colonic mucosa preparations was mounted between two halves of modified Ussing chambers, in which the total cross-sectional area was 0.5 cm 2 . The mucosal and serosal surfaces of tissue were bathed with 5 mL K-HS by recirculation from a reservoir maintained at 37 ∘ C during the experiments. The K-HS was bubbled with 95% O 2 -5% CO 2 to maintain the pH of the solution at 7.4. Drugs could be added directly to the apical or basolateral side of mucosa. Responses were continuously recorded by computer. Transepithelial potential difference for every colonic mucosa was measured by the Ag/AgCl reference electrodes (Physiologic Instruments, P2020S) connected Evidence-Based Complementary and Alternative Medicine 3 to a preamplifier that was in turn connected to a voltageclamp amplifier VCC MC6 (Physiologic Instruments). The change in SC was calculated using the value before and after simulation and was normalized as current per unit area of epithelial tissue ( A⋅cm −2 ), which allowed the curve area for 15 minutes to be calculated ( A⋅min). The change in current in response to the applied potential was used to calculate the transepithelial resistance of the monolayer by Ohm's law. Experiments were repeated in different batches of colon mucosa to ensure that the data were reproducible. Positive SC corresponded to the movement of anions from the serosal to mucosal compartments or movement of cations from the mucosal to serosal compartments or a combination of both.
2.6. Preparation of RNA and cDNA. The distal colonic, liver, and pancreas tissues were collected in phosphate buffered saline (PBS; 0.9% NaCl in 0.01 M sodium phosphate buffer, pH 7.4), which had been treated with 0.1% diethylpyrocarbonate (DEPC-PBS). After the wall of each piece of tissue had been opened, the tissue was cleaned with DEPC-PBS and transferred to Trizol (Invitrogen) for extraction of total RNA, which was isolated according to the manufacturer's instructions and stored at −80 ∘ C for later use. Samples of cDNA were generated by reverse transcription with 5 g total RNA, 50 ng random hexamer primers, and 10 nM dNTPs, incubated at 65 ∘ C for 5 min, and placed on ice for at least 1 min, with the addition of 40 URNase OUT, 200U SuperScript III RT, 10 mM dithiothreitol, and 5 mM MgCl 2 (Invitrogen), in a 20 L reaction volume. Following brief centrifugation, the reactions were incubated at 50 ∘ C for 50 min and then at 70 ∘ C for 15 min. The completed reverse transcription reactions were stored at −20 ∘ C and used for the polymerase chain reaction (PCR) without further treatment.

Statistical Analysis.
All values were expressed as means and standard error of mean (S.E.M.), and was the number of animals in each experiment. All data were analyzed using the GraphPad Prism software 5.0 package (GraphPad Software Inc., San Diego, CA, USA). The increase in SC was quantified by subtracting the peak of an SC response from its respective baseline value before drug administration. The differences between control and treatment means were analyzed using Student's paired or unpaired -test when appropriate. The differences among groups were analyzed using a one-way analysis of variance followed by Dunnett's multiple comparison. A value of less than 0.05 was considered statistically significant.

Effects of HQS, RP, and RA on the mRNA Expressions of CFTR and -ENaC.
In order to investigate whether the effects of HQS, RP, or RA on the depressed liver energy and cholestasis were related to the expressions of CFTR and -ENaC, we used real-time RT-PCR analysis to examine the expression levels of CFTR and -ENaC in liver, pancreas, and

Effects of HQS on the Protein Expression of CFTR and -ENaC.
Western blotting was performed to investigate CFTR and -ENaC protein content in gastrointestinal tract. We probed lysates from liver, distal colon, and pancreas with anti-CFTR and -ENaC antibody (Figures 2(a)-2(c)). The immunoblots were located at the same level as their corresponding positive controls. The immunoblot detected with CFTR and -ENaC antibody was 165 KD and 95 KD which was in the range reported for CFTR and -ENaC protein previously. As expected, the content levels of CFTR protein significantly increased in the liver from 0.18 ± 0.02 to 0.64 ± 0.22 ( = 6, < 0.05, about 255.5%), distal colon from 0.66 ± 0.11 to 1.05 ± 0.26 ( = 6, < 0.05, about 59.1%), and pancreas from 0.14 ± 0.02 to 0.22 ± 0.08 ( = 6, < 0.05, about 57.1%) in the HQS group more than that in the control. However, there was no significant difference in the other groups, except RA in colon group. Meanwhile, -ENaC protein contents have no significant changes in all tissues (shown in Table 2 and Figures 2(a)-2(c)).

HQS-Induced SC Responses in Rat
Model. In order to further investigate whether HQS has the choleretic action. The model rats had been administered the HQS with 8 g/kg body weight for 6 weeks. As shown in Figures 5(a) and 5(d), the baseline of SC in the model rat is 59.27 ± 6.22 A/cm 2 higher than that in the control group 47.46±6.91 A/cm 2 ( = 8, > 0.05, 19.9%), while in Figure 5(b), the transmembrane resistance in the model is 40.14 ± 2.32 Ω⋅cm 2 lower than that in the control group 49.79 ± 2.36 Ω⋅cm 2 about 19.90% ( = 8, > 0.05). As indicated in Figures 5(c) and 5(d), basolateral addition of forskolin (100 M) induced a rapid SC rise from 59.27 ± 6.22 A/cm 2 to 221.50 ± 24.29 A/cm 2 ( = 8, < 0.01) in model group, which was higher than in the control group from 45.67 ± 2.86 A/cm 2 to 107.50 ± 11.72 A/cm 2 ( = 8, < 0.01) in model group, respectively. The results indicate that the Δ SC obviously increased after forskolin application was cAMP-dependent on the CFTR expression of the apical side, while basolaterally applied bumetanide, Na + -K + -2Cl − cotransporter inhibitor (100 M), or apically applied glibenclamide (1 mM), CFTR channel inhibitor, decreased the forskolin-induced SC response to 53.65 ± 0.09 A/cm 2 ( Figure 5(d)), which was close to the baseline. hepatobiliary transporters may lead to cholestatic disorders [9]. The impaired secretory function of the biliary epithelium is considered responsible for reduced biliary fluidity and alkalinity for subsequent bile duct damage by cytotoxic compounds or infectious agents [18]. Some experiment results indicate that treatment with ursodeoxycholic acid [19], aimed at improving biliary secretion in terms of bile viscosity and bile acid composition, is currently the most useful therapeutic approach in cystic fibrosis-associated liver disease. Similarly, it was previously shown that tight junctional integrity and transepithelial resistance are relatively resistant to ischemia in bile ducts [20]. The principal effect of HQS is to regulate the functional activities of qi, eliminate stagnation of qi, and enable qi to flow smoothly [15], while Cl − channels have been shown to be important in the regulation of the hepatocyte volume in the presence of altered osmotic conditions; however, the role of this channel in bile flow has not been demonstrated [21]. Electrical parameters, which are monitored in the Ussing chambers, are widely accepted for monitoring the viability and integrity of tissue in the Ussing Chambers. SC reflects the ionic fluxes across the epithelium. In the present study, basal electrical parameters varied over a wide range. This variability has also been observed in previous studies on human tissue samples from jejunum and colon. Formation of bile requires the coordinated function of two epithelial cell types: hepatocytes that are responsible for secretion of the major osmolytes and biliary constituents and cholangiocytes that regulate the fluidity and alkalinity of bile through secretion of osmolytes such as Cl − and HCO 3 − [22]. Studies in isolated cholangiocyte preparations have elucidated the basic transport mechanisms involved in constitutive and stimulated secretory activities in the biliary epithelium. Primary damage to the biliary epithelium is the cause of several chronic cholestatic disorders [23]. From a pathophysiological point of view, common to all cholangiopathies is the coexistence of cholangiocyte death and proliferation and various degrees of portal inflammation and fibrosis. Cholestasis dominates the clinical picture and may initiate or worsen the process. Alterations in biliary electrolyte transport can contribute to the pathogenesis of cholestasis in primary bile duct diseases. Cystic fibrosis-related liver disease represents an example of biliary cirrhosis secondary to a derangement of cholangiocyte ion transport [23]. Epithelial Cl − channels play an important role in regulating and maintaining the normal physiological functions of the GI tract [24]. In this epithelium, Cl − secretion is mediated by two steps, that is, the accumulation of cytosolic Cl − by Na + -K + -2Cl − cotransporters in the basolateral membrane and then the exit of Cl − through Cl − channels in the apical membrane [25,26], namely, cystic fibrosis transmembrane regulator (CFTR). Since Cl − secretion requires both the basolateral accumulation of Cl − by Na + -K + -2Cl − cotransporter and an apical exit through Cl − channels, it is not clear at this point which site is the primary target of HQS. Our results have demonstrated that HQS ethanol extract exerted a stimulatory effect on colon mucosa Cl − secretion by predominantly activating apical cAMP-dependent Cl − channels, namely, CFTR and possibly the Na + -K + -2Cl − cotransporter [23]. The ability of HQS to stimulate Cl − secretion in the GI tract may contribute to its beneficial effects [27], such as smoothing bowl movement [28] and enhancing fluid clearance during host defense response [29]. It remains uncertain whether the stimulatory effect of HQS on Cl − secretion is due to the collective effect of all the constituent herbal components or some active ingredients contained in the ethanol extract. The general objective of the experiments was to investigate the relation between electrical activity of the intestinal epithelium and capacity to transfer fluid and various solutes. This involved the measurement of the transfer of fluid and solutes by means of short-circuit current. Nevertheless, the current study has established a model for the quantitative measurement of HQS effect on the GI tract to further investigate its possible active ingredients. This effect can potentially improve GI disorders, such as constipation, a condition commonly associated with aged people [30]. The present study demonstrated the stimulation of HQS on the colonic epithelial Cl − secretion. The supporting evidence for the stimulatory effect of HQS on Cl − secretion includes the following: (1) HQS-induced SC increase was insensitive to the Na + channel blocker amiloride but sensitive to Cl − channel blocker glibenclamide and removal of extracellular Cl − ; (2) the response was inhibited by the inhibitor of Na + -K + -2Cl − cotransporters, bumetanide. These confirmed the stimulation of Cl − secretion by HQS. Furthermore, Cl − secretion requires both the basolateral accumulation of Cl − by Na + -K + -2Cl − cotransporter and apical exit through Cl − channels [31]; (3) CFTR, but not -ENaC, in liver, pancreas, and colon, was much higher in the HQS group than those in other groups. As shown in Figures 1, 2, and 4, RA could stimulate the Cl − secretion with the enhancement of SC . Our hypothesis is supported by the observation of increased mRNA and protein of CFTR in colon and liver of the treatment group compared with controls. Using multiple methods, these data demonstrate that HQS is inherently altered in alimentary tract CFTR both in in vitro colon mucosa and in vivo models. These results suggest dominant pathway for regulation of biliary secretion to improve hepatic function [32].

Discussion
In summary, the results of this study support that HQS and RA can upregulate the expression of CFTR in alimentary tract and evoke colonic ion secretion via CFTR activation, but not -EnaC. Future studies are needed to better delineate this question.