Chunli Ma,1,* Qiqige Buren,1,* Mutu Eerde,2,* Ming Qing,3 Xiufang Bao,4 Linyun Zhao,1 Lili Cao,1 Xin Zhang,1 Qigeqi Bai,4 Meiling Chen,4 Mei Hong,1 Yulong Bao,1 Hua Lian4 

1School of Basic Medicine, Inner Mongolia Medical University, Hohhot, Inner Mongolia, 010110, People’s Republic of China; 2Medical Innovation Center for Nationalities, Inner Mongolia Medical University, Hohhot, Inner Mongolia, 010110, People’s Republic of China; 3International Mongolian Hospital of Inner Mongolia, Hohhot, Inner Mongolia, 010065, People’s Republic of China; 4College of Mongolian Medicine, Inner Mongolia Medical University, Hohhot, Inner Mongolia, 010110, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Yulong Bao; Hua Lian, Chilechuan Dairy Economic Development Zone, Hohhot, Inner Mongolia Autonomous Region, 010110, People’s Republic of China, Tel +86 18698451017 ; +86 13171029769, Email yulongbao@immu.edu.cn; lianhua81521@163.com

Propose: To elucidate the mechanisms underlying the therapeutic effects of Tonglaga-5 (TLG5) in the treatment of chronic diarrhea.
Methods: The chemical compositions of TLG5 in both in vitro and systemic circulation were analyzed using UPLC-Q/TOF MSE. Network pharmacology was applied to predict the therapeutic effects of TLG5 on chronic diarrhea. Molecular docking was utilized to assess the binding affinity between key bioactive compounds and their corresponding targets. A total of 24 male Sprague-Dawley (SD) rats were randomly assigned to three groups: control, model, and TLG5 treatment. Corresponding administration included a standard diet, a high-lactose diet of varying concentrations, and TLG5 by gavage during modeling. Transcriptomic analysis was employed to assess gene expression changes in intestinal tissues. Alterations in the intestinal microbiota were evaluated using 16S rRNA sequencing. qPCR and immunohistochemistry technologies were adopted to analyze the expression of genes and proteins associated with intestinal barrier integrity. ELISA was performed to quantify levels of bile acids and short-chain fatty acids (SCFAs).
Results: UPLC-Q/TOF MSE identified 118 compounds in vitro and 52 in the systemic circulation. Network pharmacological analysis revealed that the in vitro and in vivo components of TLG5 were associated with five core targets and five key compounds, respectively. RNA sequencing analysis showed that TLG5 treatment inhibited the inflammatory pathway. 16S rRNA sequencing demonstrated that TLG5 administration led to an upregulation of beneficial microbial populations and a concomitant downregulation of pathogenic bacteria. The TLG5 treatment group presented a reduction in necrotic areas and inflammatory cell infiltration, as well as preservation of the mucosal structure, with a marked decline in inflammatory lesions. Moreover, TLG5 treatment resulted in a significant increase in the levels of SCFAs and bile acids in the cecum.
Conclusion: TLG5 exerts therapeutic effects on chronic diarrhea through multiple mechanisms, including modulation of intestinal microbiota diversity, enhancement of intestinal barrier function, and attenuation of inflammatory responses.

Keywords: Mongolian medicine Tonglaga-5, chronic diarrhea, intestinal barrier, intestinal microbiota