| Purpose：In this research, model rats were made by multiple factors including taking exhausting anddissipating Qi medicine, adopting out of constant diet and excess fatigue which was morecorrespond with the clinic features of spleen deficiency. We tested the MUC5ACmRNA andMUC5AC protein expression, the T lymphocyte subsets and the apoptosis of rats gastricmucosa in every group to explore the relationship between the spleen deficiency and gastricmucosal barrier. This research provided the theoretical basis for treating gastric mucosaldisease by combining of Traditional Chinese Medicine and western medicine.Material and method：1. Model rats were made by multiple factors including taking exhausting and dissipating Qimedicine, adopting out of constant diet and excess fatigue, and then they were divided intodifferent groups. After one-week adaptive breeding,102healthy SD rats half male and halffemale with body weight200±10g were randomly divided into four groups,i.e.,the normalcontrol group (n=26),the long-term model group(n=26), the natural recovery group(n=26) andthe sijunzi decoction group(n=26). Rats in the normal control group were raised in the routineway for7weeks and were given normal saline1.5ml/100g by gavage once every other dayduring the first4weeks, while then became everyday in the last3weeks. Spleen deficiencyrat model was made by multiple factors in the other three groups. Model rats were made byfasting and taking exhausting and dissipating Qi medicine1.5ml/100g by gavage once everyother day and adequate feeding but swimming till endurance limit every other day. After the4-weeks’modelling time, the rats in long-term group were being modeling for another3weeks. Rats in the natural recovery group were raised in the routine way for3weeks. Rats inthe sijunzi decoction group were given sijunzi decoction1.5ml/100g by gavage onceeveryday for3weeks. All the rats were killed for drawing materials in the end of the seventhweek.3.Test the mucin changes in the rats gastric mucosa：We used Real-time PCRmethod to test the MUC5ACmRNA in rats’ gastric mucosa of every group and used western blot to test MUC5AC protein expression.4.Test the T lymphocyte subsets in the rats gastric mucosa：immunohistochemical method wasused to test the quantity change of CD4+ã€CD8+T lymphocytes and ratio change ofCD4+/CD8+in rats gastric mucosa of every group.5. Test the apoptosis of rats’ gastric mucosa: TUNEL method and immunohistochemical method was used to test the changes of apoptosis and Caspase-3in the rats’ gastricmucosa of every group.Results：1.Observing appearance and behavior of rats: rats in the long-term model group, the naturalrecovery group and the sijunzi decoction group showed a serial of appearance and behaviorchange after modeling about seven days, the hair was no luster; still, curl up, gather together,Squinting; most of them had loose or soft or wet stool, some of them had mucous stool. In thelast two weeks of modeling, they showed chaos hair, perianal filthy, several of them hadscrotal edema; most of them reduced food intake and lost weight. All of these behaviorsshowed in long-term model group from the beginning to the end, and rats in this group had asevere chaos hair, curling up and loose stool, even some of them had bloody mucopurulentstool, prolapsed, and scrotal edema. Most of those behaviors of rats in natural recovery grouphadn’t turn better after removing of modeling. Those behaviors of rats in sijunzi decoctiongroup got better after one-week time of removing modeling. At the end of experiment theappearance, exciting degree, and stool basically return to normal. Rats in normal controlgroup had no those appearance and behavior.2.The weight increase and swimming endurance: Compared with the normal control group,the mean weight increase and swimming endurance time of rats in the long-term model group,the natural recovery group and the sijunzi decoction group were all decreased, while there wasno significant difference between sijunzi decoction group and the normal control group afterpairwise comparison(P＜0.05). The mean weight increase and swimming endurance time ofrats in the long-term model group and the natural recovery group had significant differencecompared with the normal control group, while the mean weight increase of this two groupshad no significant difference after pairwise comparison(P＜0.05). 3.Spleen index and thymus index of rats: Compared with the normal control group, the spleenindex and thymus index of the long-term model group and the natural recovery groupsignificantly decreased(P＜0.05),while the spleen index of the sijunzi decoction group had nosignificant difference compared with the normal control group(P＞0.05). Compared with thesijunzi decoction group, the spleen index of the long-term model group significantlydecreased(P＜0.05),while the natural recovery group had no significant difference(P＞0.05).Compared with the normal control group，only thymus index of the long-term model groupsignificantly decreased(P＜0.05), the spleen index of the natural recovery group and thesijunzi decoction group had no significant difference(P＞0.05). Compared with the naturalrecovery group, the other groups all had no significant difference(P＞0.05).4. MUC5ACmRNA and protein expression in the gastric mucosa: Compared with the normalcontrol group, MUC5AC protein expression in both the long-term model group and thenatural recovery group were all decreased(P＜0.05),while the sijunzi decoction group had nosignificant difference(P＞0.05). Although the MUC5AC protein expression in the naturalrecovery group were higher than the long-term model group(P＜0.05), it was still lower thanthe normal control group and the sijunzi decoction group(P＜0.05). The expression ofMUC5ACmRNA of the natural recovery group was higher than the long-term model group(P＜0.05), while there was no significant difference between the natural recovery group andsijunzi decoction group(P＜0.05).5. T lymphocyte subsets in the rats gastric mucosa: The quantity of CD4+T lymphocytes inthe sijunzi decoction group couldn’t returned to the level of the normal control group(P＜0.05),while the quantity of CD8+T lymphocytes in this group had no significant differencecompared with the normal control group(P＜0.05). Compared with the long-term modelgroup and the natural recovery group, the quantity of CD4+T lymphocytes in the sijunzidecoction group increased significantly, while the quantity of CD8+T lymphocytes decreasedsignificantly(P＜0.05). Compared with the normal control group, the ratio of CD4+/CD8+inthe sijunzi decoction group, the natural recovery group, and the long-term model groupsuccessively decreased(P＜0.05).Although the ratio increased significantly in the sijunzidecoction group, it still lower than the normal control group(P＜0.05).6. The apoptosis of rats gastric mucosa: Compared with the normal control group, the apoptosis of gastric mucosa epithelial cell of the other three groups were all increased,especially the long-term model group(P＜0.05). After the treating of sijunzi decoction, theapoptosis of gastric mucosa epithelial cell descreased, it had significantly differencecompared with the long-term model group and the natural recovery group(P＜0.05).Compared with the normal control group, the Caspase-3in rats’ gastric mucosa of the othergroups were all increased(P＜0.05). After the treating of sijunzi decoction, the Caspase-3inrats’ gastric mucosa descreased, it had significantly difference compared with the long-termmodel group and the natural recovery group(P＜0.05).Conclusion：1. Spleen deficiency model rats made by multiple factors including taking exhausting anddissipating Qi medicine, adopting out of constant diet and excess fatigue have greater stability.This kind of spleen deficiency model was more in line with the essence of spleen deficiency,because the appearance and behavior of rats couldn’t recover by themselves after the removeof modeling.2.The decrease of mucin MUC5AC,the quantity of CD4+T lymphocytes, the ratio of CD4+/CD8+, and the increase of the quantity of CD8+T lymphocytes, Caspase-3and apoptosis inspleen deficiency model rats gastric mucosa all prompted the damage of the gastric mucusbarrier, Mucosal immune barrier, Mucosal repair function and the weakened of gastricmucosa barrier function.3. Strengthening spleen and nourishing Qi could improve the function of gastric mucosabarrier by increasing the mucin, adjusting the T lymphocytes subsets and decreasing theapoptosis, which proved that spleen deficiency could weaken the function of gastric mucosabarrier and there should be certain correlation between the spleen deficiency and gastricmucosa barrier function. |
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