The anti diarrheal effect of different polarity parts of windproof on diarrhea rats and the regulation of different subtypes of AQP in the colon
Diarrhea is a digestive system disease caused by multiple pathogens and factors, resulting in increased frequency, thin or watery stool, often accompanied by clinical symptoms such as abdominal pain, vomiting, fever, and varying degrees of fluid and electrolyte imbalance. Epidemiological studies have shown that the global incidence of diarrhea is about 5% per year, and it is also the second leading cause of death in children under 5 years old. The intestine is the main site for water transport and metabolism, with approximately 9L of water secreted and absorbed in the intestine every day. It is of great significance for maintaining water balance and digestive absorption function in the body. The decrease in water reabsorption in the intestine is an important cause of diarrhea. The colonic reabsorption of water is a reverse gradient active absorption process, mainly relying on the transport function of aquaporins (AQPs). AQP is the main protein responsible for the transmembrane transport of water molecules, which can efficiently and selectively transport water molecules to maintain the water liquid balance of the intracellular and extracellular environment. At least 11 different subtypes of AQP are expressed in the intestine, which not only serve as important carriers for the absorption and metabolism of intestinal water molecules, but also participate in the secretion of intestinal mucus and nutrient solution, as well as the maintenance of the homeostasis of the intestinal intracellular and extracellular environment. They play an important role in regulating some intestinal nerve functions. Abnormal expression of AQP in the intestine can cause disturbances in the intestinal environment, leading to reduced absorption of water molecules and a large accumulation of water molecules in the intestinal lumen, resulting in the occurrence of diarrhea. With the continuous deepening of research on AQP, it is expected to become a target for the prevention and treatment of many intestinal diseases related to water metabolism and transport.
Diarrhea belongs to the category of “diarrhea” in traditional Chinese medicine. Traditional Chinese medicine believes that “if dampness prevails, diarrhea will occur” and “if there is no dampness, diarrhea will not occur”. Dampness is the key to the pathogenesis of this disease. Feng medicine is a type of medication that has the effects of dispelling wind and dampness, promoting hair growth and clearing yang. Its pungent and warm properties have a significant therapeutic effect on diarrhea. Windproof is a typical representative of wind medicine, which is the dried root of Saposhnikovia di-varicata (Trucz.) Schischk., a plant in the Umbelliferae family. It has the effects of dispelling wind, relieving surface dampness, and relieving pain. It is also used for diarrhea caused by spleen deficiency, dampness, and yang deficiency due to its ability to clear and dry dampness. The clinical efficacy of windproof treatment for diarrhea is definite, but its effect on intestinal AQP in diarrhea rats has not been reported. In this study, a rat model of diarrhea was established by gavage of senna leaves. The effects of windbreak and its different polar extracts on the body mass, diarrhea index, electrolytes, and diamine oxidase of diarrhea rats were observed, DAO)、Na+-K+-ATPase、 The effects of tumor necrosis factor – α (TNF – α) and the protein expression of AQP3, AQP4, AQP-8, and AQP-9 in colon tissue are investigated to explore the possible mechanism of action of windbreak and antidiarrheal drugs. This will help expand the clinical application of windbreak drugs and provide reference for the development of related drugs.

Diarrhea is an intestinal disease caused by various factors that lead to a decrease in the absorption or secretion function of water in the intestine, resulting in an increase in the water content of feces. It belongs to the category of “diarrhea” in traditional Chinese medicine, and its pathogenesis is spleen deficiency and excessive dampness, dysfunction of transportation and metabolism, resulting in the inability to distinguish between clear and turbid, and the infiltration of water and dampness, leading to diarrhea. Traditional Chinese medicine believes that when dampness prevails, it leads to dampness and release, while wind energy triumphs over dampness. Feng medicine belongs to the category of pharmacology with the function of dispelling wind and dampness, promoting hair and clearing yang, and is a type of medication used for pathogenic wind diseases. Wind medicine has a light qi and a thin taste, with a strong and pungent aroma. Due to its warm and dry nature, it can dispel dampness. AQP is a protein family that specifically transports water across membranes, involved in water absorption and transport, and closely related to the generation, distribution, and excretion of bodily fluids. Traditional Chinese Medicine believes that the pathogenic factors of dampness can be divided into visible and tangible dampness diseases such as edema and diarrhea, and potentially invisible dampness diseases such as wind dampness and rheumatism, but both are related to the imbalance of body fluids. AQP may be the biological basis for the occurrence of body fluid diseases. Based on the traditional Chinese medicine theory of “wind energy triumphs over dampness”, AQP may be a target for wind medicine treatment. Windbreak is a representative medicine of wind medicine, used to treat diarrhea due to its ability to promote clearing and dry dampness. Therefore, clarifying the antidiarrheal effects of different polarity parts of windproof and their impact on colonic AQP can help expand the clinical application of windproof drugs and provide reference for the development of related drugs.
Senna leaf is a commonly used laxative, mainly composed of senoside, emodin, aloe emodin, and rhein. It can increase intestinal peristalsis, inhibit the digestion and absorption of water and other substances in the intestine, stimulate the synthesis and release of inflammatory mediators in the intestine, damage the intestinal mucosa, and promote intestinal emptying. Therefore, it is commonly used as a model drug for diarrhea animal models in pharmacological research due to its significant laxative effect. This study showed that by the 5th day of oral administration of senna leaves to rats, the diarrhea rate had reached 100%; On the 7th day, the body mass of the model rats decreased significantly compared to normal rats, and the diarrhea index increased significantly. After stopping the oral administration of senna leaves in rats with diarrhea caused by senna leaves, it is possible to restore normal function. Therefore, in this experiment, senna leaves were continued to be orally administered on the 8th day to eliminate the effect of automatic recovery of animal diarrhea symptoms. After the end of administration, the body weight of rats in the Difenofibrate group, Windbreak petroleum ether group, n-butanol group, and total extract group increased compared to the model group, and the diarrhea index decreased, indicating that Windbreak has a good anti diarrheal effect. The petroleum ether and n-butanol parts may be its effective anti diarrheal parts.
Research on the pathogenesis of diarrhea has found that pathogenic factors activate adenylate cyclase, which converts intracellular ATP into cAMP. The activity of colon Na+- K+- ATPase decreases, promoting the efflux of K+from intestinal mucosal cells and the entry of Na+, Cl -, and other substances into the cells, causing swelling and necrosis of the cells. The water content in the intestinal lumen increases significantly, leading to diarrhea and electrolyte imbalance. DAO is a highly active intracellular enzyme present in intestinal mucosal epithelial cells. When intestinal mucosal cells are damaged, DAO releases human blood, leading to an increase in serum DA0 activity. Therefore, the activity of DAO can reflect the degree of intestinal mucosal damage and evaluate the status of intestinal mucosal barrier function. When diarrhea occurs, the intestinal mucosa is damaged, macrophages and T cells are activated, promoting the synthesis and release of TNF – α. TNF – α further promotes the secretion of inflammatory factors (such as IL-1, IL-6, etc.), exacerbating intestinal mucosal damage, causing edema, congestion, and forming a vicious cycle. This study showed that the concentrations of Na+, K+, and Cl – in the serum of rats with diarrhea induced by senna leaf were significantly reduced, the levels of DAO and TNF – α were increased, the activity of Na+- K+- ATPase in colon tissue was decreased, and pathological morphology showed damage to colon mucosa. The concentrations of Na+, K+, and Cl – in the serum of rats were increased to varying degrees in the Difenofibrate group, Windproof n-butanol group, Petroleum Ether group, and Total Extract group. The levels of DAO and TNF – α were decreased, and the activity of Na+- K+- ATPase in colon tissue was increased. The damaged colon mucosa was repaired to varying degrees, indicating that Windproof Petroleum Ether group, n-butanol group, and Total Extract can repair damaged intestinal mucosa and balance electrolyte imbalances.
The intestine is an important organ for water transport and metabolism in the body. In the early days, it was believed that water transport in the intestine depended on the osmotic pressure difference inside and outside the intestinal lumen produced by electrolytes. Now, research has found that the colonic reabsorption of water is an active absorption process mediated by AQP reverse osmosis gradient. The abnormal expression of AQP in the intestine is a key factor leading to reduced water absorption and subsequent accumulation of large amounts of water, which is closely related to the imbalance of the intestinal environment. At present, 13 subtypes of aquaporins (AQPO~AQP12) have been identified, of which 11 are expressed in the intestine, with AQP3, 4, 8, and 9 playing the main roles. AQP3 is a relatively selective constitutive aquaporin that can transport glycerol and urea in addition to water. Studies have found that AQP3 regulates the fecal water content in the colon by mediating the rotation of water between the intestinal lumen and blood vessels; AQP4 is mainly located in the basal lateral membrane of colonic epithelial cells. As a highly selective aquaporin, it has the ability to rapidly transport water, which is 3-4 times more permeable than other aquaporins. It transports water molecules from the intestinal lumen to the intestinal interstitium and plays an important role in the digestive system; AQP8 is expressed in the mucosal absorption epithelial cells of the colon, participating in the reabsorption of water by the colon. Its expression is significantly reduced in the colon of patients with diarrhea, and the lower the expression of AQP8 in the colon, the higher the fecal water content; AQP9 not only participates in colonic fluid metabolism, but also synthesizes and secretes mucus, protects intestinal mucosa and lubricates stool, which is associated with the occurrence of constipation. This experiment showed that the expression of AQP3, 4, and 8 in the colon of diarrhea model rats was downregulated, while AQP9 showed no significant change, indicating the reabsorption basis of AQP on intestinal water; Difenofibrate, petroleum ether fraction, n-butanol fraction, and total extract can increase the protein expression of AQP3, AQP4, and AQP-8 in colon tissue of diarrhea rats; It is worth noting that the components of Difenofibrate and Windproof Petroleum Ether can reduce the levels of AQP9 in the colon of rats, while the total extract of Windproof has no significant effect on AQP9. It is speculated that other parts of Windproof may regulate the abnormal expression of AQP9. The above results indicate that Windproof and its petroleum ether and n-butanol fractions may exert antidiarrheal effects by regulating the expression of colonic AQP, and petroleum ether and n-butanol fractions have different effects on the regulation of different subtypes of AQP.
In summary, windbreak has a good anti diarrheal effect, which may be related to increasing Na+- K+- ATPase activity, repairing mucosal damage, and regulating intestinal AQP expression. The petroleum ether and n-butanol parts are the active sites of windbreak and anti diarrheal, and they have different effects on AQP expression in different subtypes of the intestine. The regulation mechanism of AQP varies among different subtypes, and the differential regulation mechanism of AQP by windbreak and its different polar parts needs further research.