| Background and purpose:Our previous data showed that licorice water extract and licorice miRNA156 had certain impacts on intestinal microorganisms and immune system of healthy mice,but it was not further explored whether licorice and licorice mirna156 had regulatory effects on mice in the disease model.This study was first carried out from the traditional therapeutic efficacy,we used chronic fatigue model mice(spleen deficiency)as study object to explore the effect of treatment of chronic fatigue model mice by licorice extract and miRNA156 extract,and their impact on the intestinal flora and immune system.Method:1.In the first two weeks,we adopted the method of combined stress stimulation which involved sleep deprivation and forced swimming to establish the model of chronic fatigue syndrome in mice.During the third week,intervention treatment was carried out.Mice were divided into four groups,the model group was gavaged with normal saline,Ge group with licorice water extract,mimic group with licorice miRNA156 mimic,mut group with licorice miRNA156 mutant.The body weight was recorded every day,and the tail suspension test and stress pain test were carried on days 1,14,and 21.Second day after the intervention experiment,behavioral experiments were conducted and after anatomy,we collected samples for testing fatigue related index,and complete ileal tissues of mice were taken to prepare Swiss rolls to prepare slices for HE staining analysis..2.After the intervention,the feces of mice were collected for 16 S high-throughput sequencing,and the effects of intervention treatment on intestinal microorganisms of mice in each group were analyzed.3.Based on the transcriptome data previously,ten differential genes were selected as the genes ready to be tested according to the pathway of significant difference.After the interference experiment,the total RNA and protein of mice ileum tissue were extracted to carry out qPCR and westing blotting assays.4.After interference treatment,part of the mice spleen tissue was fixed into sections for HE staining analysis and immunohistochemistry;Spleen cells were extracted from the remaining spleens,flow cytometry and qPCR assay were carried out respectively.Results:1.In the model group,the fatigue mice showed weight loss,mental depression,hair loss,dyspepsia,and the decreasing ability of action response,space independent exploration and environmental adaptation,gradually;The weight and excitement of mice in Ge group and mimic group increased,which could significantly improve the ability of autonomous activity and adapting to the environment,and reduce malondialdehyde and TNF-α expression;There was no significant difference in liver index;Cecal index in Ge group was similar to that in con group,and the index increased in other groups;The spleen weight and the number of spleen cells were significantly down-regulated in each exercised mice.HE staining showed that the intestinal tissue structure of the mice in the model group was severely damaged,resulting in limited intestinal digestion and absorption function and affecting the physiological state of the body;after treatment with licorice water extract and miRNA156,we clearly saw that the shape of villi was repaired.It indicated that licorice and its miRNA156 could effectively relieve the related symptoms and repair the intestinal structure of fatigued mice.2.The results of 16 S high-throughput sequencing showed that the microbial diversity and abundance of mice in the con group and other groups were significantly different;the diversity and abundance of mice intestinal flora in the group treated by licorice water extract were close to the con group;Licorice water extract mainly regulates Bacteroides and Lactobacillus,while miRNA156 mainly regulates Lactobacillus;through functional prediction,it was found that licorice water extract treatment can relieve fatigue symptoms in mice by regulating immunity and nerve cells,miRNA156 mainly effected detoxification and metabolism.3.The results of qPCR showed that under different physiological conditions,the qPCR results of ileum had some opposite regulatory trends with the previous transcriptome data.The expression trend of mCd19,mCd79 a,mCxcr4,and mCxcr5 is the same as that of the previous data,and the expression of mPtprc has the opposite trend;the licorice water extract and its miRNA did not significantly regulate mCd28,mCr2,and mCxcl12;in the mimic group,mCd4 and mTgfbr2 were not regulated at all effect.The expression of CD19 molecule in ileal tissue of licorice water extract group at the protein level was consistent with its expression trend at the m RNA level.4.The results of spleen slices showed that long-term fatigue can lead to a severe ratio imbalance in terms of white pulp to red pulp in the spleen of mice,white pulp suffered a severe loss and its edges were blur,which seriously disrupts the immune system homeostasis of the spleen,resulting in a series of spleen deficiency symptoms in mice;the licorice water extract group had a significant improvement,the proportion of white pulp area increased and the edge was clear,and the miRNA156 group also showed therapeutic effect.qPCR results showed that GE group and mimic group affected the expression of Cd19,Cd28,Cd79 a,Cxcr5,Ptprc and other genes.The results of flow cytometry and immunohistochemistry showed that the licorice water extract group had stronger and more stable regulation of mCD19 expression than after miRNA156 intervention.Conclusion:When mice experience high-intensity exercise and mental stress for a long time,they will experience chronic fatigue syndrome symptoms such as weight loss,lethargy,hair loss,and the ability to explore and adapt to space autonomously,which will lead to damage in intestinal structure and affect the body digestive function.Furthermore this damage can also cause a serious imbalance in the structure of intestinal flora which leads to depletion of the body’s immunity;after treatment with licorice water extract and miRNA156,the damaged part of the body’s intestinal tract is repaired,inflammation is down-regulated,and at the same time,it promotes the restoration of intestinal microbial diversity balance,and the spleen structure also recovered to a certain extent,the expression levels of immune genes in the gut and spleen also changed,and multiple symptoms of chronic fatigue syndrome were alleviated. |
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