| Background:"Sphincter of Oddi(SO)dysfunction(SOD)is a functional disorder caused by SO dysfunction,leading to abnormal bile or pancreatic juice flow.SOD primarily manifests as abdominal pain,bloating,nausea,and jaundice.It’s understood that SOD is not only a significant factor in the pathogenesis of most biliary and pancreatitis-related functional diseases,but it’s also a key secondary pathological change in these diseases.However,its specific pathogenesis remains unclear.Therefore,it’s imperative to explore the precise pathogenesis of SOD and find more effective and reliable treatment methods.Studies indicate that interstitial cells of Cajal not only maintain basic rhythm pacing and spreading function but are also primary target cells of intestinal nerve function.The excessive apoptosis of interstitial cells of Cajal plays a critical role in the onset and development of SOD.Clinical acupuncture has been used for pain relief for thousands of years,and electroacupuncture,as a straightforward method of acupuncture treatment,has a positive regulatory effect on SO motor function.However,the basic experimental study on the impact and mechanism of electroacupuncture on SOD has not been reported.This study confirms that hypercholesterolemia in rabbits is a significant model of SOD.Objective:To explore the effect of electroacupuncture on the motility of the Oddi sphincter and the apoptosis of interstitial cells of Cajal in the Oddi sphincter in hypercholesterolemic rabbits.The aim is to reveal the mechanism of electroacupuncture on Oddi sphincter dysfunction in hypercholesterolemic rabbits,thereby providing a theoretical basis for acupuncture treatment of clinical Oddi sphincter dysfunction diseases.Method:Twenty-four healthy rabbits were randomly divided into three groups:normal control group(6 rabbits),hypercholesterolemia model group(6 rabbits),and treatment group(ST36,6 rabbits in Zusanli group and ST36+rabbits in Zusanli and its acupointmatching period and Yanglingquan group,6 rabbits).The normal control group was fed with standard feed,while the high cholesterol model group and the treatment group were fed with high cholesterol feed.Eight weeks after the model was established,200 uL of blood was collected from each rabbit’s ear vein,and total serum cholesterol was measured.Total serum cholesterol exceeding 10 mmol/L indicated successful model construction.Then,electroacupuncture treatment was applied.The ST36 treatment group received electroacupuncture stimulation at bilateral Zusanli points in rabbits,and the ST36+treatment group received electroacupuncture stimulation at bilateral Zusanli points in rabbits,simultaneously matched with bilateral Qimen points and Yanglingquan points.After 34 days(that is,after three treatment courses),SO tissues and serum were collected.SO tissue morphology was observed through a microscope,SO pressure was measured to assess SO function,the levels of IL-6,IL-1β,TNF-α,and oxidative stress factor SOD in serum were detected,and the changes in mRNA levels of SCF,STAT3,and JAK2 in SO tissues of rabbits in each group were analyzed by PCR technology.The expressions of c-kit protein,Bcl-2,Bax,and Cleaved capase3 apoptosis-related proteins in the Oddi sphincter were analyzed by immunohistochemistry,and the expression levels of c-kit protein,Bcl-2,Bax,and Cleaved capase3 apoptosis-related proteins in the Oddi sphincter were analyzed by Western Blot.The apoptosis of c-kit protein in Oddi sphincter was detected by TUNEL staining and immunofluorescence staining.Results:1 Model Evaluation:The serum total cholesterol level in the hypercholesterolemia model group and the electroacupuncture treatment group was significantly higher than that in the normal control group(>10 mmol/L)(p<0.0001).This indicates that the hypercholesterolemia rabbit model was successfully established.The successful establishment of the hypercholesterolemia rabbit model was further verified by the pathological morphology and pressure test results of the Oddi sphincter tissue in the hypercholesterolemia model group after HE staining.2 HE Staining:The tissue morphology of the Oddi sphincter in the model group was damaged to varying degrees compared to the normal control group.In the high cholesterol model group,the tissue structure in SO was chaotic;the glands in the muscle layer were irregularly arranged,the gap was widened,the arrangement was loose,and there was infiltration of inflammatory cells.After electroacupuncture treatment,the pathological morphology of the rabbits in the electroacupuncture treatment group improved,with a relatively tighter tissue structure compared to the high cholesterol group.3 SO Pressure Measurement:The SO pressure in the high cholesterol model group significantly increased,and the contraction amplitude decreased compared to the normal control group.After electroacupuncture treatment,the SO basal pressure in the electroacupuncture treatment group significantly decreased,and the contraction amplitude increased compared to the rabbits in the hypercholesterolemia model group.4 Changes in Inflammatory Factors and Oxidative Stress Factors in Rabbit Serum:The contents of inflammatory factors such as TNF-α,IL-6,and IL-1β in the serum of rabbits in the hypercholesterolemia model group significantly increased,while the sod content significantly decreased compared to normal control rabbits.After electroacupuncture treatment,the contents of TNF-α,IL-6,and IL-1β inflammatory factors in the electroacupuncture treatment group decreased significantly,while the sod content increased significantly.5 Apoptosis Results of Interstitial Cells of Cajal:TUNEL and c-kit immunofluorescence double staining showed that the apoptosis of c-kit-labeled interstitial cells of Cajal in high cholesterol model rabbits increased compared to normal control rabbits,while apoptosis decreased after electroacupuncture treatment.Using the immunohistochemical method and WB to detect changes in c-kit,Bcl-2,Bax,and Cleaved capase3 protein expression levels in SO tissues showed that the expression levels of c-kit protein and Bcl-2 protein in high cholesterol model rabbits decreased,while the expression levels of apoptosis-related proteins of Bax and Cleaved capase3 increased.In the electroacupuncture treatment group,the expression levels of c-kit and Bcl-2 protein in SO tissue of rabbits increased,while the expression levels of Bax and Cleaved capase3 protein decreased.6 Detection of SCF,STAT3,and JAK2 mRNALevels in SO Tissue:The expressions of SCF,STAT3,and JAK2 mRNA in SO tissue of rabbits in the high cholesterol model group significantly decreased compared to the normal control group.After electroacupuncture treatment,the expressions of SCF,STAT3,and JAK2 mRNA in SO tissue of rabbits significantly increased compared to those in the hypercholesterolemia model group.Conclusions:1 The level of total serum cholesterol has a significant impact on the motility of the rabbit’s Sphincter of Oddi(SO),with a noticeable increase in SO pressure in hypercholesterolemic rabbits.After electroacupuncture treatment in hypercholesterolemic rabbits,the SO pressure significantly decreases and the motility of the SO improves.This indicates that electroacupuncture has a good therapeutic effect on Sphincter of Oddi Dysfunction(SOD)in hypercholesterolemic rabbits.2 The method of electroacupuncture treatment can reduce the apoptosis of Cajal interstitial cells in the SO of hypercholesterolemic rabbits.The electroacupuncture treatment method regulates the apoptosis of Interstitial Cells of Cajal(ICC)through the SCF/c-kit and JAK2/STAT3 signaling pathways.The electroacupuncture treatment method regulates the apoptosis of ICC by modulating the expression levels of apoptosis proteins Bax,Bcl-2,and Cleaved caspase-3. |
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