Dioscin ameliorates slow transit constipation in mice by up-regulation of the BMP2 secreted by muscularis macrophages

Document Type : Original Article


1 Department of Pediatric Surgery, General Hospital, Tianjin Medical University, Tianjin, China

2 Department of General Surgery, General Hospital, Tianjin Medical University, Tianjin, China


Objective(s): The loss of enteric neurons has been shown to be a major cause of slow transit constipation (STC). Gut microbiota and muscularis macrophages (MMs) are associated with the enteric nervous system (ENS) development and gastrointestinal (GI) motility. This study aimed to investigate whether Dioscin (DIO) increased GI motility and inhibited neuron loss by modulating gut microbiota profile, improving inflammation in the ENS microenvironment.
Materials and Methods: The STC model was established by loperamide. The alteration of the gut microbiota was analyzed by 16S rDNA sequencing. The longitudinal muscle and myenteric plexus (LMMP) from the colon were prepared for flow cytometry, immunofluorescence, western blot, and qRT-PCR. 
Results: DIO increased the stool number, stool water content and shortened whole gut transit time, helped to recover the gut microbial diversity and microbiota community structure, and increased the abundance of Muribaculaceae in STC mice. Compared with the STC group, the number of MMs and the level of the iNOS, IL-6, and TNFα genes were significantly decreased following DIO treatment. Moreover, DIO may increase the number of HuC/D+ neurons per ganglion by up-regulating the BMP2 secreted by MMs and activating the BMP2/p-Smad1/5/9 signaling pathway. Furthermore, the level of excitatory neurotransmitter AchE in colon tissues exhibited a substantial increase in the DIO group. However, the level of inhibitory neurotransmitter VIP was markedly decreased. 
Conclusion: Our results provide that DIO increases GI motility and inhibits neuron loss by modulating gut microbiota profile, improving inflammation in the ENS microenvironment and up-regulating the BMP2 secreted by MMs.


1. Guerin A, Mody R, Fok B, Lasch KL, Zhou Z, Wu EQ, et al. Risk of developing colorectal cancer and benign colorectal neoplasm in patients with chronic constipation. Aliment Pharmacol Ther 2014; 40: 83-92.
2. Sumida K, Molnar MZ, Potukuchi PK, Thomas F, Lu JL, Yamagata K, et al. Constipation and risk of death and cardiovascular events. Atherosclerosis 2019; 281: 114-120.
3. Stern T, Davis AM. Evaluation and treatment of patients with constipation. JAMA 2016; 315: 192-193.
4. Muller PA, Koscso B, Rajani GM, Stevanovic K, Berres ML, Hashimoto D, et al. Crosstalk between muscularis macrophages and enteric neurons regulates gastrointestinal motility. Cell 2014; 158: 300-313.
5. Becker L, Nguyen L, Gill J, Kulkarni S, Pasricha PJ, Habtezion A. Age-dependent shift in macrophage polarisation causes inflammation-mediated degeneration of enteric nervous system. Gut 2018; 67: 827-836.
6. Liu W, Zhang Q, Li S, Li L, Ding Z, Qian Q, et al. The relationship between colonic macrophages and microrna-128 in the pathogenesis of slow transit constipation. Dig Dis Sci 2015; 60: 2304-2315.
7. Bassotti G, Villanacci V, Maurer CA, Fisogni S, Di Fabio F, Cadei M, et al. The role of glial cells and apoptosis of enteric neurones in the neuropathology of intractable slow transit constipation. Gut 2006; 55: 41-46.
8. Liu X, Liu S, Xu Y, Liu X, Sun D. Bone morphogenetic protein 2 regulates the differentiation of nitrergic enteric neurons by modulating Smad1 signaling in slow transit constipation. Mol Med Rep 2015; 12: 6547-6554.
9. Serra J, Pohl D, Azpiroz F, Chiarioni G, Ducrotte P, Gourcerol G, et al. European society of neurogastroenterology and motility guidelines on functional constipation in adults. Neurogastroenterol Motil 2020; 32: e13762.
10. Nijenhuis CM, ter Horst PG, van Rein N, Wilffert B, de Jong-van den Berg LT. Disturbed development of the enteric nervous system after in utero exposure of selective serotonin re-uptake inhibitors and tricyclic antidepressants. Part 2: Testing the hypotheses. Br J Clin Pharmacol 2012; 73: 126-134.
11. Roerig JL, Steffen KJ, Mitchell JE, Zunker C. Laxative Abuse : epidemiology, diagnosis and management. Drugs 2010; 70: 1487-1503.
12. Yao Y, Cui L, Ye J, Yang G, Lu G, Fang X, et al. Dioscin facilitates ROS-induced apoptosis via the p38-MAPK/HSP27-mediated pathways in lung squamous cell carcinoma. Int J Biol Sci 2020; 16: 2883-2894.
13. Cai S, Chen J, Li Y. Dioscin protects against diabetic nephropathy by inhibiting renal inflammation through TLR4/NF-kappaB pathway in mice. Immunobiology 2020; 225: 151941.
14. Qi Y, Li R, Xu L, Yin L, Xu Y, Han X, et al. Neuroprotective effect of dioscin on the aging brain. Molecules 2019; 24:1247.
15. Li C, Lu Y, Du S, Li S, Zhang Y, Liu F, et al. Dioscin exerts protective effects against crystalline silica-induced pulmonary fibrosis in mice. Theranostics 2017; 7: 4255-4275.
16. Rtibi K, Grami D, Selmi S, Amri M, Sebai H, Marzouki L. Vinblastine, an anticancer drug, causes constipation and oxidative stress as well as others disruptions in intestinal tract in rat. Toxicol Rep 2017; 4: 221-225.
17. Jeon JR, Lee JS, Lee CH, Kim JY, Kim SD, Nam DH. Effect of ethanol extract of dried Chinese yam (Dioscorea batatas) flour containing dioscin on gastrointestinal function in rat model. Arch Pharm Res 2006; 29: 348-353.
18. Wahba G, Hebert AE, Grynspan D, Staines W, Schock S. A rapid and efficient method for dissociated cultures of mouse myenteric neurons. J Neurosci Methods 2016; 261: 110-116.
19. Joseph NM, He S, Quintana E, Kim YG, Nunez G, Morrison SJ. Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut. J Clin Invest 2011; 121: 3398-3411.
20. Gabanyi I, Muller PA, Feighery L, Oliveira TY, Costa-Pinto FA, Mucida D. Neuro-immune interactions drive tissue programming in intestinal macrophages. Cell 2016; 164: 378-391.
21. Yarandi SS, Kulkarni S, Saha M, Sylvia KE, Sears CL, Pasricha PJ. Intestinal bacteria maintain adult enteric nervous system and nitrergic neurons via toll-like receptor 2-induced neurogenesis in mice. Gastroenterology 2020; 159: 200-213 e208.
22. Chandrasekharan B, Saeedi BJ, Alam A, Houser M, Srinivasan S, Tansey M, et al. Interactions between commensal bacteria and enteric neurons, via FPR1 induction of ROS, increase gastrointestinal motility in mice. Gastroenterology 2019; 157: 179-192 e172.
23. He Q, Han C, Huang L, Yang H, Hu J, Chen H, et al. Astragaloside IV alleviates mouse slow transit constipation by modulating gut microbiota profile and promoting butyric acid generation. J Cell Mol Med 2020; 24: 9349-9361.
24. Jiang H, Dong J, Jiang S, Liang Q, Zhang Y, Liu Z, et al. Effect of durio zibethinus rind polysaccharide on functional constipation and intestinal microbiota in rats. Food Res Int 2020; 136: 109316.
25. Yi R, Peng P, Zhang J, Du M, Lan L, Qian Y, et al. Lactobacillus plantarum CQPC02-Fermented Soybean Milk Improves Loperamide-Induced Constipation in Mice. J Med Food 2019; 22: 1208-1221.
26. Shang L, Liu H, Yu H, Chen M, Yang T, Zeng X, et al. Core altered microorganisms in colitis mouse model: A comprehensive time-point and fecal microbiota transplantation analysis. Antibiotics (Basel) 2021; 10:643.
27. Kim JE, Park JW, Kang MJ, Choi HJ, Bae SJ, Choi YS, et al. Anti-Inflammatory Response and Muscarinic Cholinergic Regulation during the Laxative Effect of Asparagus cochinchinensis in Loperamide-Induced Constipation of SD Rats. Int J Mol Sci 2019; 20:946.
28. Ren X, Liu L, Gamallat Y, Zhang B, Xin Y. Enteromorpha and polysaccharides from enteromorpha ameliorate loperamide-induced constipation in mice. Biomed Pharmacother 2017; 96: 1075-1081.
29. Wu M, Li Y, Gu Y. Hesperidin improves colonic motility in loeramide-induced constipation rat model via 5-hydroxytryptamine 4R/cAMP signaling pathway. Digestion 2020; 101: 692-705.
30. Wehner S, Behrendt FF, Lyutenski BN, Lysson M, Bauer AJ, Hirner A, et al. Inhibition of macrophage function prevents intestinal inflammation and postoperative ileus in rodents. Gut 2007; 56: 176-185.
31. Yang M, Fukui H, Eda H, Kitayama Y, Hara K, Kodani M, et al. Involvement of gut microbiota in the association between gastrointestinal motility and 5HT expression/M2 macrophage abundance in the gastrointestinal tract. Mol Med Rep 2017; 16:3482-3488.
32. Anitha M, Shahnavaz N, Qayed E, Joseph I, Gossrau G, Mwangi S, et al. BMP2 promotes differentiation of nitrergic and catecholaminergic enteric neurons through a Smad1-dependent pathway. Am J Physiol Gastrointest Liver Physiol 2010; 298:G375-383.
33. Saglam A, Kim S, Ahn K, Oh I, Lee KH. BMP2 shows neurotrophic effects including neuroprotection against neurodegeneration. Neuroreport 2014; 25: 549-555.
34. Song NN, Lu HL, Lu C, Tong L, Huang SQ, Huang X, et al. Diabetes-induced colonic slow transit mediated by the up-regulation of PDGFRalpha(+) cells/SK3 in streptozotocin-induced diabetic mice. Neurogastroenterol Motil 2018;30:1-14.
35. Feldman M, Schiller LR. Disorders of gastrointestinal motility associated with diabetes mellitus. Ann Intern Med 1983; 98: 378-384.
36. Honore SM, Zelarayan LC, Genta SB, Sanchez SS. Neuronal loss and abnormal BMP/Smad signaling in the myenteric plexus of diabetic rats. Auton Neurosci 2011; 164: 51-61.
37. Lee JI, Park H, Kamm MA, Talbot IC. Decreased density of interstitial cells of Cajal and neuronal cells in patients with slow-transit constipation and acquired megacolon. J Gastroenterol Hepatol 2005; 20: 1292-1298.
38. Hwang DY, Kim S, Hong HS. Substance-P ameliorates dextran sodium sulfate-induced intestinal damage by preserving tissue barrier function. Tissue Eng Regen Med 2018; 15: 63-73.
39.Huizinga JD, Tomlinson J, Pintin-Quezada J. Involvement of nitric oxide in nerve-mediated inhibition and action of vasoactive intestinal peptide in colonic smooth muscle. J Pharmacol Exp Ther 1992; 260: 803-808.