Functional constipation is one of the main subtypes of irritable bowel syndrome [8]. Clinically, persistent or indirect abdominal pain and abnormal stool trait symptoms caused by gastrointestinal peristalsis disorder, intestinal secretion disorder and other factors, and more are the results of the interaction of brain-gut axis disorder, visceral sensory abnormalities, abnormal gastrointestinal motility, intestinal flora imbalance and other related factors [9]. In recent years, due to the influence of diet and other factors, the incidence of functional constipation in Asia is increasing year by year, because of its long duration, easy to cure a lot of inconvenience to people's life [10]. Liushenqu was first published in Medicine, which is a mixture made of flour, wheat bran, bitter almonds, red beans, artemisia annua, polygonum and xanora. It is rich in vitamin B complex, digestive enzymes, volatile oils, ergosterols, glycosides and flavonoids. Is widely used in the treatment of various gastrointestinal diseases, [11].
Numerous studies have proved that the brain-gut peptides, widely distributed in the brain and the gastrointestinal tract, have the interaction between connecting and regulating various links of the brain-gut axis, including participating in regulating the movement, sensation and secretion of the gastrointestinal tract, and have the dual effects of neurotransmitter and hormone [12, 13].There are dozens of intestinal peptides, among which 5-HT and VIP are important excitatory and inhibitory peptides in gastrointestinal motility, respectively, and the disorder of both levels can induce intestinal dysfunction.[14]
VIP is a peptidergic neurotransmitter, mainly distributed in the mucosal lamina propria and myometrial nerve fibers, which can regulate intestinal motility and secretion activities, and is closely related to the formation of constipation. VIP is mainly distributed in the human gastrointestinal tract, which can relax the smooth muscle of the gastrointestinal tract and affect the gastrointestinal peristalsis of [15]. Lu et al [16]. found higher VIP levels in the STC rat model group than in the normal group, indicating that the increased concentration of VIP can inhibit gastrointestinal smooth muscle movement. However, Giancola et al and [17] found that the number of submembrane neurons in constipation was decreased, and the expression of VIP and VIPR1 was significantly reduced compared with normal people. Although the results of the VIP level study are opposite, the reduced VIP level is not contradictory with the slowing of colonic movement. When the intestinal content is advanced in the intestine, the proximal intestinal tube contraction and the distal intestinal tube relaxation are needed to make the stool through smoothly [18]. As for the increased level of VIP in STC patients, some scholar believes that VIP can produce nitric oxide by promoting the synthesis of target cells, so the increase of VIP level can promote nitric oxide to increase intestinal motility and relax the smooth muscle [19] .
At present, the mechanism of VIP relaxation of gastrointestinal smooth muscle is that the combination of VIP and specific receptor produces cascade amplification effect [20], Ca2 + influx and activation endothelial nitric oxide synthase (eNOS), leading to increased NO synthesis and activation of protein kinase G (PKG), which causes smooth muscle relaxation. Activated nitric oxide synthase (NOS) -positive neurons are heavily expressed, produce a persistent effect on inhibiting intestinal contraction, leading to from constipation[21, 22] .VIP neurons are an important part of the submucosa, and their abnormal secretion affects the composition of fecal water content, leading to difficult stool and delayed colonic transmission [23, 24]. Studies have shown that the Liushenqu contains a large number of monomer components such as quercetin, oxin, isoxin, and these components have good antioxidant activity and can directly regulate the level of VIP, which is directly related to the increase of VIP level in serum [25].
The human gut is a huge and complex microecosystem. Through the interaction, bacteria participate in the growth and development of the body and maintain the balance and the stability of the body. The occurrence and development of many gastrointestinal diseases are also directly or indirectly related to the change of microflora [26]. The analysis of common species in this experiment showed that the number of unique OTUs in the model group increased slightly (compared with the CC group), and the unique OTUs increased sharply after the treatment. The differential analysis of family level bacteria showed that the proportion of Lactobacillus in CM group was significantly lower than that in CC group, and the proportion of harmful bacteria increased significantly, and the structure of six flora was closer to that of CC group, suggesting that Liushenqu can treat functional constipation by regulating the structure of intestinal flora.
As a medicine for stomach digestion, Liushenqu has been used in clinical practice since ancient times, especially rice pasta. Zhao et al. [27] analyzed the changes of gastrin, cholinesterase and NO in serum content in mice after filling Liushenqu, and found that the secretion of gastrin and cholinesterase in mice increased, and the content of NO in serum decreased. Wang et al. [28] studied the antibacterial effect of the ethyl acetate, and found that the minimum inhibitory concentration was 0.64 gL− 1 and the lowest bactericidal concentration was 0.65 gL− 1. Through experimental research, Guo et al [29] destroyed the intestinal microbial flora of mice, formed intestinal tissue ulcers, and filled the liquid. Using natural recovery as the control, the intestinal ulcers in the experimental group recovered quickly, which also confirmed the effect of Liushenqu on the repair of intestinal ulcers in mice. Liu et al. [30] analyzed the contractile effect of Liushenqu on the rat intestinal smooth muscle, and found that Liushenqu could enhance the contraction effect of the rat intestinal smooth muscle. Zhuang et al [31] compared the number changes of fecal Lactobacillus and bifidobacterium and enterobacteria in patients with irritable bowel syndrome (IBS). The results showed that the number of beneficial bacteria in the feces increased after taking the drug, and the growth of enterobacteria was inhibited. Liushenqu is rich in digestive enzymes. In the fermentation process, the fermentation matrix of special microorganisms or microbes is decompose to produce amylase and protease, etc., and then achieve the effect of healthy stomach and digestion.
Short-chain fatty acids (SCFAs) mainly includes formate, acetate, propionate and butyrate, etc. They are formed by the fermentation of indigestible carbohydrates by the intestinal flora. SCFAs not only provide important nutrients and energy for enterocytes, but are also precursors of adipogenesis and gluconeogenesis. These short-chain fatty acids play important roles in energy metabolism, immunity, and adipose tissue expansion[32]. SCFAs mainly regulate intestinal function in the following ways: inhibition of histone deacetylase (HDAC) to regulate gene expression, and combined with SCFAs receptors to affect intestinal function. The identified SCFAs receptors include:G protein-coupled recep-tors (GPCR) 41,43 and 109A (GPR 41, GPR 43, GPR109A), which are found over intestinal epithelial cells, immune cells, endocrine cells and adipocytes, and are closely related to intestinal metabolism, tumor suppression and immune regulation[33].The SCFAs in the gut can not only be absorbed as nutrients but also regulate intestinal function. The metabolite SCFAs will affect the fecal transport time of the colon, which is negatively correlated[34]A large number of animal experiments, tissue in vitro experiments Confirmed that SCFAs is involved in regulating intestinal motility and can improve constipation symptoms[35–38].Studies have shown that butyrate can significantly increase the proportion of acetylcholine transferase (ChAT) immunoreactive positive interneurons and can increase the cholinergic-mediated isolated colonic cricoid contraction response[39]The main effect of butyrate occurs after it is transported intracellular by the monocarboxylate transporter (MCT) belonging to the proton-linked membrane proteins of the Slc16a1 family. MCT has now been found in mammals, 14 members of the family, but among these only MCT 1, MCT 2, MCT 3, and MCT 4 have been shown to transport monocarboxylates such as lactate, pyruvate, β -hydroxybutyrate, or SCFA. Butyrate increases the colonic motor and contractile responses induced by the ENS via the cholinergic pathway, increasing the proportion of ChAT-IR enteric neurons.The butyrate-induced cholinergic phenotype involves MCT 2, which is specifically detected in enteric nerve cells.Butyrate has a regulatory effect on the transcription of ChAT genes and can increase both ChAT activity and mRNA stability. Butyrate can regulate gene expression by inhibiting the histone deacetylase (HDAC) in the promoter region of the gene.Ono et al. [13] found that SCFAs could affect the spontaneous contraction of longitudinal muscle (LM) in the distal part of the rat colon, including mucosa, and propionate and butyrate could increase the frequency of spontaneous contraction of LM in the distal colon of the rat. The study found that SCFAs may regulate the frequency of spontaneous contraction of LM through ENS, thus promoting colonic movement including peristaltic reflex[40].
The increase of VIP can promote the expression of various anti-inflammatory factors, such as IL-4, IL-10, and TGF- β[41, 42], Play the role of regulating inflammation, maintain the balance of pro-inflammatory and anti-anti-inflammatory cytokines, regulate the proliferation and repair of intestinal mucosal epithelium by promoting epidermal growth factor secretion, and play a protective role in the intestinal mucosal barrier[43]In addition, VIP can also improve the intestinal mucosal tissue microcirculation and internal environment, provide nutrients and oxygen for intestinal epithelial cells, and remove excessive oxygen free radicals and other harmful substances[44], reduce the damage of the intestinal mucosa by LPS, and promote the colonization of beneficial bacteria to normalize the proportion of bacterial flora, and further improve functional constipation.
In conclusion, the intestinal microflora imbalance in patients with functional constipation, mainly manifested by the decrease in the number of probiotics and the increase in the number of harmful bacteria, and the sharp decrease in the serum VIP level. By regulating the structure of intestinal flora, Liushenqu affects the secretion of short chain fatty acids in the intestinal tract and regulates the intestinal function, while short chain fatty acids stimulates the increase of the expression of VIP. VIP further regulates the intestinal flora through its own action, forming a virtuous cycle, so as to achieve the effect of relieving functional constipation.