| Background: Syndrome of deficiency of spleen-Qi(SDSQ) is the basic syndromedifferentiation of viscera in Trational Chinese Medicine. It is mainly reflected in thedigestion, absorption of abnormal process, loss of appetite, abdominal distention aftereating, stool pond thin, poor appetite. It is often caused by improper diet, overstrain,eating cold and chronic gas consumption. The main clinical manifestations of SDSQ areabdominal pain, diarrhea, common in edema, phlegm, asthma, flaccidity, stomachache,rickets in children as well as the western medicine to chronic gastritis, peptic ulcer, theulcerous colitis, chronic bronchitis, bronchial asthma and other diseases. In recent years,the study found that the patients of SDSQ suffered from dysfunction of gastrointestinalmotility and acupuncture at Zusanli(st36) and Tianshu(s25) can effectively treat thepatients with SDSQ, but the detailed mechanism was not detected.Objective: To make the model of syndrome of deficiency of spleen-Qi in rats inducedwith the method of bitter cold diarrhoea and consumption, observing the morphologicalchanges of ENS and the smooth muscle cells of small intestine in SDSQ and thetherapeutic effects of acupuncture at Zusanli, Tianshu.Methods:Sixty healthy adult Wistar rats weighing200~250g, half male and halffemale,were randomly divided into control group (10rats), SDSQ group (10rats),spontaneous recovery group (10rats), acupuncture Zusanli treatment group (Zusanli) (10rats）, acupuncture treatment group Tianshu (10rats), acupuncture Zusanli andTianshu treatment group(10rats). Each rats of control group was fed with free food and4ml distilled water for30days; SDSQ group, Zusanli treatment group, Tianshutreatment group,Zusanli and Tianshu were given party①and side②alternate lavage[①: rhubarb, magnolia bark, acid-insoluble ash (2:1:1) made from200%concentrationdecoction;②: the broad bean root, inula flower, betel nut decoction (3:1:1) made from200%concentration], first20days,3times/day, last15days,4times/day,4ml each time.When the model was established successfully, the rat of the treament group was givenacupuncture by needle at their scheduled points for2weeks by Hua Tuo HANS-200Eacupuncture therapeutic apparatus,initial current stimulate increased to0.5mA to1.5mA, daily morning7to10PM; Meanwhile, the rats of spontaneous recovery groupwere fed as normal. The proximal segment of jejunum was taken and studied using themethods below:(1) HE staining: to make good specimens of paraffin embedding,sectioning, HE staining, then to observe the morphological changes of small intestinetissue under the microscope;(2) Immunofluorescence staining: using a capsulevesicular acetylcholine transporter (VAChT) antibodies in the small intestine ofcholinergic nerve fibers single taged for fluorescence staining; With PGP9.5antibodiesand alpha actin for double marking of small intestine smooth muscle cell fluorescencestaining and laser scanning confocal microscope, the image was analysed and detected,observed between groups in the rat small intestine morphological structure of neuralnetwork and smooth muscle cells.(3) Tunel apoptotic test: observation by using TunelApoptosis Technique in the rat small intestine smooth muscle and the number ofapoptotic cells was detected.Results:Macropathology Compared with those in control group, the gastrointestinaltracts were distended significantly in the rats with SDSQ group. Compared with SDSQgroup, the changes in rats with recovery group was slightly better. Compared with theSDSQ group and recovery group, the damages in rats with acupuncture alone Zusanli orTianshu and both of them was markedly alleviated. Compare with acupuncture aloneZusanli, acupuncture alone Tianshu showed no significant difference. Micropathlogy in HE Staining Control group: the complete intestinalstructure was clear with uniform distribution of muscle layers of cells. SDSQ groups:There were many places of the small intestinal mucosa defect or broken, while epithelialcells were flaking and the intestinal villi appears to be edema. Recovery groups: It canbe occasionally seen that the small intestinal mucosa burst, however, the muscular layerstructure was clear. The small intestinal villus edema was still apparent withinflammatory cells infiltrated and part of the intestinal mild hyperemia. Zusanli groups:Compared with recovery group, the small intestinal mucosa with occasional burst,epithelial cells distributed equably, muscular layer structure was clear, fluffy mildedema. Tianshu groups: Compared with recovery group, small intestinal mucosa wasbroken, and muscle layer of the organization structure was clear, villous edema was notobvious. Compared with Zusanli group, we could see no significant difference. Zusanliand Tianshu groups: basic complete intestinal mucosa, the epithelial cells weredistributed evenly, muscular layer structure was clear, villus edema was obviouslyalleviate, the priorities of cells was neat and no obviously shorten of the small intestinalmucosa, there was a decrease in the number of nucleus, inflammatory cells infiltration,visible part adenomas had mild hyperemia.Immunofluorescence①The small intestinal nerves: Compared with thecontrol group, the quantity and the integrity optical density(IOD) of cholinergicnerves(Ach) in SDSQ group was significantly decreased(P<0.01), the nerve networkwas significantly disrupted. Compared with SDSQ group, the quantity and the IOD ofcholinergic nerves(Ach) in recovery group were better(P<0.01), the nerve network wasslightly recovered. Compared with recovery group, the number of Ach nerves in Zusanlior Tianshu groups were significantly increased(P<0.01), the quantity and the IOD ofcholinergic nerves(Ach) in Zusanli or Tianshu group were significantlyincreased(P<0.01). The nerve network was slightly recovered. Compared with recoverygroup, the number of Ach nerves in Zusanli and Tianshu groups were significantlyincreased(P<0.01), the quantity and the IOD of cholinergic nerves(Ach) in Zusanli andTianshu group were significantly increased(P<0.01). The nerve network was significantly recovered. Compared with Zusanli or Tianshu group, the number of Achnerves in Zusanli and Tianshu groups were significantly increased(P<0.01),the quantityand the IOD of cholinergic nerves(Ach) in Zusanli and Tianshu group were significantlyincreased(P<0.01).②Intestinal nerve and muscle network structure: Compared withthe control group, the number of SDSQ intestinal nerves and smooth muscle cells of ratssignificantly reduced, the IOD value of intestinal nerves was significantly lower(P<0.01), loose connections, network structure between nerve and muscle were obviousdamaged; Compared with SDSQ group, the number of rat intestinal nerve of recoverygroup were slightly increased, and the number of small intestine smooth muscle alsoslightly increased, but there was not obvious difference. Compared with the recoverygroup, the number of smooth muscle and nerves of intestine were incresed, and theconnection between intestinal nerves and muscle were more closely in Zusanli group,the network-like structure was relatively clear, and the IOD of intestinal nervesincreased. Compare with the recovery group, the number of intestinal nerve and muscleof Tianshu group increased, the mutual connection between the two was also moreclosely, intestinal nerve-muscle network-like structure was clearer, intestinal nerveIOD was increased (P<0.01). Compare with the recovery groups, the mutual connectionbetween nerves and smooth muscle of Zusanli and Tianshu groups was significanttighter, the net-like structure was significantly clearer, the IOD also increased obviously(P<0.01).③The account of apoptotic Smooth muscle cell: Compared with the controlgroup, the number of apoptotic cells in SDSQ group of rats was significantly increased(P<0.01), the complete network-like structure disappeared; compared with SDSQ group,the number of apoptotic cells of recovery group in rats relatively decreased (P<0.01),but was still more than the treatment groups (P<0.01); the number of apoptotic cells inZusanli group was significantly less than in recovery group (P<0.01); number ofapoptotic cells in rats treated by acupuncture in Tianshu was significantly reduced(P<0.01); No significant difference between Zusanli group and Tianshu group (P>0.01).Conclusions:①The gastrointestinal tracts of the rats with SDSQ induced by bitter colddiarrhoea and qi consumption were significantly distension, and the syndrome of gastrointestinal obstruction and syndrome of enteroparalysis were significant.②HEstaining showed that rat intestinal of SDSQ group has great damage in microscopicpathology, Zusanli or Tianshu acupuncture can improve their pathologic damage,acupuncture Zusanli and Tianshu can significantly improve the damage of pathologycaused by SDSQ.③The number of cholinergic nerves(Ach) were reduced in SDSQ.The nerve network was disrupted in SDSQ significantly. Acupuncture Zusanli andTianshu could prevent the decreasing of the enteric nerve and repair the enteric nervenetwork.④Acupuncture Zusanli and Tianshu could eventually suppress the apoptosisof smooth muscle of intestine,so that it could repair the damage and improve thefunction of digestive system. |
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