| Background Syndrome of Deficiency of spleen-Qi (SDSQ) is a specific term inChinese traditional medicine, the symptoms include anorexia, abdominal distension andloose stools. As the point of view of modern medicine, SDSQ is mainly characterized asdeficiency of gastrointestinal absorption and motility dysfunction. The motility functionof the gastrointestinal tract is an important physiological function of digestive system,therefore the recovery of gastrointestinal motility function is the first and critical step toremedy the damage of gastrointestinal dysfunction. Studies has shown that theinterstitial cells of Cajal (interstitial cells of Cajal, ICC) were widely distributed in thegastrointestinal tract, which was a class of special cells between enteric nervous system(ENS) and smooth muscle cells (SMC). The enteric nervous releases the relativeneurotransmitter on the ICC, which integrates the information and acts on smoothmuscle cell so that causing rhythmically gastrointestinal movement. Enteric nervous,ICC, and smooth muscle cells are connected to each other, which constitute the networkstructure together as the basic functional unit of gastrointestinal motility (BFUGM). TheBFUGM is closely related to gastrointestinal motility dysfunction. Studies found thatthe gap junction (GJ) existed among ICCs, between ICC and SMC and between SMCs.Connexin43(Cx43) is the most important protein in connections protein family, whichit is the hub of integrating GJ information. It plays an essential role in maintaining thejunction of the intercellular migration signal transduction and rhythmic contractions ofthe smooth muscle. And the normal expression of the protein is the substructure ofgastrointestinal motor function. It is closely related with the gastrointestinal motility.Objective To make the model of SDSQ in rats and observe the morphologicalchanges in network of ICC-SMC, using the confocal laser scanning microscope and transmission electron microscope. Observe the distribution of connexin43and thequantity and structure changes of gap junction in muscular layer of gastrointestine in ratwith SDSQ, using the immunohistochemistry and transmission electron microscope.The mechanism of gastrointestinal motility disorders and the therapeutic effects ofXiang-Sha-Liu-Jun-Zi decoction (XSLJZD) would be explored.Methods Sixty healthy adult Wistar rats of both sexes weighing180to220gwere randomly divided into three groups: control group (n=20), SDSQ group(n=20) andtreated group(n=20). The model of SDSQ were made by alternately gavage withprescription A (raw rhubarb, citrus aurantium, magnolia,200%concentration decoction)and prescription B (sophora root, inula, betel,250%concentration decoction) for30days(the first18days, the prescription A or B was gavaged for three times a day; thefollowing12days, it was gavaged for four times a day). According to the rats in SDSQmodel group, the normal saline was gavaged for the rats in control group. After themodel was successfully established, the XSLJZD (2ml/100g weight day) was gavagedfor14days for the rats of treated group. In the rats of control group and SDSQ modelgroup, the normal saline was gavaged for14days according to the rats in XSLJZDtreated group. The immunohistochemical method, transmission electron microscopymethod, laser scanning confocal microscopy were used, and the morphological changesin network of ICC-SMC and gap junction of small intestine and colon in rats withSDSQ, and therapeutic effects of XSLJZTD were analyzed.Results Anatomical changes Compared with those in control group, the intestinaltracts were more significantly distended and intestinal obstruction was in the rats ofSDSQ group. Compared with the SDSQ group, the changes in rats in XSLJZD groupwas markedly alleviated.Immunofluorescence Network of ICC-SMC in small intestine and colonBeforethe SDSQ model was established, the number of ICC and integrated optical density(IOD) value of ICC and SMC were analyzed. There were no statistically significanceamong the three groups. After the rat model with SDSQ was successfully established,the number of ICC and IOD value of ICC and SMC were significantly decreasedcompared with the control group(P<0.05). On the14th day after XSLJZTD was used,the number of ICC and IOD value of ICC and SMC in treated group were significantlyincreased compared with the SDSQ group(P<0.05). There was no statisticallysignificance between the control group and treated group in the number of ICC and IODvalue of ICC and SMC. On the day when the SDSQ model was established, the number of ICC and IOD value of ICC and SMC were significantly decreased (P<0.05)compared with that before the model established. On the14th day after XSLJZTD wasused, the number of ICC and IOD value of ICC and SMC were significantly increased(P<0.05) compared with that on the day when the SDSQ model established.Immunohistochemistry There was positive staining of Cx43protein in controlgroup which was brownish yellow sediment, it was densely distributed throughout thecell membrane. Different tissues had different distribution. The Cx43protein of smallintestine distributed mainly in the whole circular muscle layer, particularly around theouter edge of the circular muscle layer, and it was also found between the circularmuscle layer and longitudinal muscle layer in colon.Before the SDSQ model established, the IOD of expression of Cx43was analyzed.There were no statistically significant among the three groups. After the rat model withSDSQ was successfully established, the IOD of expression of Cx43was significantlydecreased compared with the control group(P<0.05). On the14th day after XSLJZTDwas used, the IOD of expression of Cx43in treated group was significantly increasedcompared with the SDSQ group (P<0.05). There was no statistically significant betweenthe control group and treated group to the IOD of expression of Cx43. On the day whenthe SDSQ model was established, the IOD of expression of Cx43was significantlydecreased (P<0.05) compared with that before model established. On the14th day afterXSLJZTD was used, the IOD of expression of Cx43was significantly increased(P<0.05)compared with that on the day when the SDSQ model established.Transmission electron microscopy ICC was characterized by large, oval,light-staining nuclei with little perinuclear cytoplasm, from which the2-5primary cellprocesses were extended, giving the cells a spindle shape or a stellate configurationbefore the SDSQ model established. The primary processes were connected withprocesses of neighboring ICC and SMC. The gap junctions were between adjacent cellmembranes. After the rat model with SDSQ was successfully established, comparedwith the control group, the nuclei of ICC in SDSQ were shrunken. Injury was displayedin both perinuclear and processes. Mitochondria was decreasing and swelling. The gapjunctions between ICCs, ICC and SMC and between SMCs were significantlydecreased. On the14th day after XSLJZTD was used, compared with the SDSQ group,the nuclei, perinuclear cytoplasm and mitochondria in treated group were remained, andthe gap junctions between ICCs, ICC and SMC and between SMCs were significantlyincreased. Conclusions The intestinal mucosa layer of the SDSQ rats were congestion andedematous, and colonic obstruction was obvious; XSLJZD could alleviate thecongestion and edematous, restore the intestinal normal form.The number of ICC in small intestine and colon was greatly reduced in SDSQgroup, network structure was damaged; XSLJZD could protect the structure damage ofICC in SDSQ rat, so that the network structure was protected.The expression and distribution of Cx43in small intestine and colon were greatlyreduced in rat with SDSQ; XSLJZD could protect the structure damage of Cx43in ratof SDSQ.The quantity of GJ in network of ICC-SMC were reduced in rats with SDSQ, andthe structure was damaged; XSLJZD could maintained the quantity of GJ, protect thestructure of GJ from destructing, so that maintained the structural and functionalintegrity in gastrointestinal network of ICC-SMC. |
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