| Background: Sphincter of Oddi (SO) whose end is connected with the majorduodenal papilla is a sphincter independent from the duodenal wall muscle layer. SO isthe only gateway for discharge of bile from the biliary duct. SO regulates the secretionof bile and pancreatic juice, and maintains the stability of the low-pressure and low-flowsystem in the biliary duct[1-2]. SO is considered as the "gate" for the flow of pancreaticjuice and bile, and is the most important anatomic structure and functional area in thecholedocho-pancreatico junction. Sphincter of oddi dysfunction (SOD) refers to thecontractile dysfunction of SO and is most commonly manifested by sphincterhypertonia. SOD is one benign noncalculous obstruction where the bile and pancreaticjuice pass through the choledocho-pancreatico junction. SOD includes movementdysfunction and structural changes of SO, and these lesions can harm bile sphincteror/and pancreatic sphincter. At present, the curative methods of SOD include endoscopy,surgery, and medication, but no effective drug is specific for SO. Therefore, it is urgentto find Chinese and Western medicines for treatment of SOD, and to improve thediastolic function of SO.Wei Jingguo proved that hypercholesterolemia (HC) indeed could induce SOD, butthe specific mechanism is unknown[3]. The occurrence of SOD was proved to be relatedto the overload of calcium ions in SO cells. Calcium ions are the second messenger incells, and the variation of intracellular calcium ion level ([Ca2+]i) is closely related tothe contractile function of smooth muscle. Ion channels are pervasive in membranes ofsmooth muscle cells. These proteins are directly involved in the formation andregulation of intracellular calcium ion levels, where potassium ion channels play veryimportant roles. Voltage-dependent potassium channel (Kv) and large conductanceCa2+-sensitive K+channels (BKca) are the most abundant and pervasive in smoothmuscle cell membranes, and have high effect on membrane potential[4-6]. They become the effective targets of many drugs on SOD, especially diastolic dysfunction of cardiacmuscles.Shaoyao Gancao Decoction was introduced by Zhang Zhongjing in Treatise onFevers, and is composed of peony and Zhigancao. Shaoyao Gancao Decoction is acommonly used analgesic method, and experiments showed its sedative, analgesic, andsmooth muscle-relaxing effects. Reportedly, Shaoyao Gancao Decoction had relaxingeffects on the movement of in-vitro rabbit intestinal tubes[7]and precontracted rat womb.We used peoniflorin extracted from the Chinese traditional medicine Radix PaeoniaeAlba to treat rabbit SO rings in-vitro. The results showed that peoniflorin had evidentrelaxing effect on Ach precontracted SO rings. However, the mechanism for the relaxingeffect of Shaoyao Gancao Decoction on SO has not been reported. Therefore, in thispaper, RT-PCR and Western Blotting were used to observe the changes of gene andprotein expression in Kv4.3channel and BKca channel in SO cells of HC rabbits, whichreceived Shaoyao Gancao Decoction of different doses. We aimed to investigate themechanism and thus to provide some foundation for the clinical application of ShaoyaoGancao Decoction in treatment of SOD.Objectives:1. To observe the effects of HC on the mRNA and protein expressions inKv4.3channel, BKca channel α subunits, and BKca channel β1subunits in rabbit SOcells.2. To investigate the effects of different doses of Shaoyao Gancao Decoction on themRNA and protein expressions in Kv4.3channel, BKca channel α subunits, and BKcachannel β1subunits in HC rabbit SO cells.Methods:1. Health clean-bred New Zealand rabbits were randomly divided into5groups: control group, HC group, high-dose group, mid-dose group, and low-dose group.The control group was fed with standard rabbit stuff; the HC group and the three dosegroups were fed with standard stuff+cholesterol. The rabbits were raised for12weeks,and after induction of HC, the three dose groups underwent intragastric administrationof Shaoyao Gancao Decoction for4weeks, while normal saline was used instead in theother two groups.2. SO tissues were collected from each group for RT-PCR and Western Blotting,followed by photograph by using gel imaging system. The mRNA and proteinexpressions in Kv4.3channel, BKca channel α subunits, and BKca channel β1subunitsin rabbit SO cells were analyzed. The grey value ratio of aim strips to GAPDH strips was used to express the relative expression level of mRNA in the aim channel; the ratioof grey value of aim channel strip to the optical density of β-actin strip was used toexpress the relative expression level of the corresponding protein in the aim channel.Results:1. HC evidently reduced the mRNA and protein expressions in Kv4.3channel, but did not affect the mRNA and protein expressions of BKca channel αsubunits and BKca channel β1subunits.2. Shaoyao Gancao Decoction could apparently upregulate the mRNA and proteinexpressions in Kv4.3channel, BKca channel α subunits and BKca channel β1subunitsin HC rabbits. This effect was dose-dependent, and was the largest in the high-dosegroup and the smallest in the low-dose group. The low-dose Shaoyao Gancao Decoctionshowed no apparent upregulative effect on the mRNA and protein expressions in Kv4.3channel.Conclusions:1. The variation of protein expression in Kv4.3channel in SO cellsmay be one cause for the downregulated activity of K+channel in HC rabbits. ShaoyaoGancao Decoction had relaxing effect on SO by up-regulating the protein expression inKv4.3channel.2. HC did not apparently induce the expression changes in BKca channel α subunitsand BKca channel β1subunits, but Shaoyao Gancao Decoction indeed could cure SODthrough upregulating the expression in BKca channel.3. The upregulative effects of Shaoyao Gancao Decoction on the mRNA and proteinexpressions in Kv4.3channel, BKca channel α subunits, and BKca channel β1subunitsin HC rabbits were dependent on doses, and the large dose showed the highest effect. |
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