Ruscogenin Alleviates Intestinal Bleeding and Blood Flow Induced by Dasatinib through ROCK/MLC Pathway

Background As a second-generation broad-spectrum tyrosine kinase inhibitor, Dasatinib has the antitumor effect of inhibiting tyrosine kinases such as BCR-ABL and SRC. It is mainly used to treat chronic and acute patients with chronic myelogenous leukemia. However, Dasatinib has many side effects, including gastrointestinal bleeding, respiratory infections, and renal failure, and effective means to improve the side effects of drugs are lacking. Methods C57BL/6 mice and HUVECs were used to evaluate the side effects caused by dasatinib, RUS was administered in advance to prevent, protein expression and phosphorylation were measured by western blot, and monitored the intestinal bleeding with Laser Doppler Blood Flow Monitor. We found that Ruscogenin (RUS) can improve Side effects caused by Dasatinib. Through the Laser Doppler Blood Flow Monitor, it was found that after Dasatinib was administered, blood ow in mice are decreased, and intestinal Evans blue leakage increased. Western blot results showed that connexin (ZO-1, VE-cadherin, Occludin) is destroyed, the activation of ROCK increases, and the phosphorylation of MLC increases. Ruscogenin can reverse the above phenomenon and improve the side effects of gastrointestinal bleeding caused by Dasatinib. In vitro, giving Ruscogenin in advance can protect the endothelial barrier damage caused by Dasatinib, reduce the remodeling of F-actin, and restore the expression of connexin (ZO-1, VE-cadherin), inhibit the activation of ROCK, and reduce Phosphorylation of MLC. This study provides a new direction for improving the side effects caused by clinical drugs and conrm that Ruscogenin could the side effect from Dasatinib by ROCK/MLC pathway.


Background
In clinical treatment, drug-induced side effects not only aggravate the patient's condition, but also bring a great nancial burden to the patient [1] . A survey data shows that 71.3% of patients who require hospitalization due to acute bleeding caused by drugs [2] . Therefore, prevention and treatment of side effects caused by drugs have a positive effect on the treatment of patients.
As a second-generation broad-spectrum tyrosine kinase inhibitor, Dasatinib exerts an anti-tumor effect by inhibiting tyrosine kinases such as BCR-ABL and SRC [3][4] . Clinically usemainly in the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia [5] , as well as leukemia patients resistant to Imatinib, Dasatinib inhibits wild-type BCR-ABL much stronger than the rst-line treatment drug Imatinib At 325 times [6][7] . Dasatinib's global sales in 2016 were $1.8 billion. Although Dasatinib is very effective, it often shows serious side effects. The most common side effects in clinical reports include gastrointestinal symptoms [8] , hemorrhagic events, hepatotoxicity [9] , pleural effusion [10][11][12] , pulmonary hypertension [13][14] , and respiratory infections with neutral granules. Adverse events such as cell reduction and renal failure [15] .
Clinical investigations shown that 23% of patients taking Dasatinib have developed bleeding, and gastrointestinal bleeding is a relatively high incidence of adverse reactions. Among patients with gastrointestinal bleeding, most patients received high doses of Dasatinib (> 100 mg) or received twice daily doses [16][17][18] . It can be judged that oral Dasatinib can cause gastrointestinal bleeding. The possible mechanism of Dasatinib's destruction of the endothelial barrier is due to the activation of ROCK / MLC signals, which causes actin polymerization and the breakdown of connexins in vascular endothelial cells.
Intervention in the ROCK / MLC signaling pathway has been recognized for protecting the blood-brain barrier [19][20] .Therefore, intervention of the endothelial ROCK-MLC signaling pathway and improvement of barrier disorders is to alleviate Dasatinib-induced intestinal bleeding.
At present, there are not many effective monitoring methods to examine the patient's bleeding.Some Articles have shown that an increase in organ bleeding can cause a decrease in blood ow [21][22] .Most of the models used to study the side effects caused by Dasatinib are intraperitoneal injection or oral lowdose long-term administration [15,[23][24] . Animal models of large-dose oral administration are lacking. In clinical use, large-dose oral administration is also common. Therefore, large-dose oral administration was selected.
Ruscogenin is one of the effective active ingredients of Ophiopogon japonicus, which has effect on antiin ammatory, anti-thrombosis and reduces capillary permeability [25][26] . Ruscogenin can alleviate endothelial cell injury, protect blood-brain barrier function, and relieve pulmonary hypertension caused by monocrotaline [27][28] .In the early stage of the laboratory, It was proved that Ruscogenin can up-regulate the expression of adhesion proteins VE-cadherin and p120-catenin, inhibit the nuclear translocation of VEcadherin, thereby improving the adhesion of endothelial cell adhesion junction and reducing the damage of the pulmonary vascular endothelium induce by Lipopolysaccharide [29] .
Based on the above data, we hypothesized that RUS can be used as a clinical intervention to improve the side effects of Dasatinib causing endothelial dysfunction.

Animals
All animal care and experimental procedures were carried out according to the current European Communities Council-ECC guidelines for the care of laboratory animals and ethical guidelines for investigations of experimental pain in conscious animals. Animal studies were reported in compliance with the ARRIVE guidelines. Male C57BL/6J mice (20-25 g; 8 weeks; speci ed pathogen free) were provided by the Model Animal Research Centre of Yangzhou University (Yangzhou, Jiangsu, China) and kept in cages containing standard bedding, with at least ve mice per cage. Mice were housed in a speci c pathogen-free facility with 12 h light/dark cycle [07 to 19 h, temperature (22 ± 2°C), humidity (40-70%), controlled ventilation] and with sterile water and irradiated food available ad libitum. Animals were allowed to acclimatize to their housing environment for at least 7 days prior to experimentation and to the experimental room for 1 h before experiments.

Drug treatments
Ruscogenin was weighed and dissolved in absolute ethanol to prepare a mother liquor. When used, the corresponding concentration was adjusted with physiological saline. Dasatinib was dissolved in physiological saline at a concentration of 140 mg/kg and administered intragastrically.30 minutes after the administration of Ruscogenin, Dasatinib was administered by gavage.After 30 minutes, the mice were killed.

Blood ow monitoring
Thirty minutes before administering Dasatinib, mice were given Ruscogenin, and 2 hours later anesthetized with Pelltobarbitalum Natricum. The small intestine was removed and placed on a Doppler owmeter for scanning to obtain images.

Western blot analysis
Western blotting analysis was performed as previously described. The cells was lysed and centrifuged.

Histology and immunohistochemistry
The excised vessel was dehydrated with 40% sucrose, embedded in optimal cutting temperature (OCT) and frozen at −70°C. The jejunum was sectioned into slices of 10 μm of thickness with a cryotome (Leica, Mannheim, Germany). Specimens were washed in PBS and stained with haematoxylin and eosin (H&E).
For immunohistochemical staining, slides were incubated with primary antibodies at 4°C overnight. Alexa Fluor 488-/594-labelled antibodies were used as secondary antibodies. All results are expressed as the means±SD. Statistical analysis was performed using Student's two-tailed t test for comparison between two groups and one-way analysis of variance (ANOVA) followed by Dunnett's test when the data involved three or more groups. P<0.05 was considered statistically signi cant. All analyses were performed using GraphPad Prism Version 5.01 (GraphPad Software Inc.USA).

Oral Dasatinib reduced intestinal blood ow in mice
Clinically, Dasatinib is used for oral treatment of chronic myeloid leukemia. In this section, the time points of blood ow reduction in the small intestine caused by Dasatinib were observed by different doses of Dasatinib (70, 140, 210 mg/kg). As shown in Fig.1, administration of 70 mg/kg Dasatinib did not caused evident decrease blood ow in intestinal . Compared with the control group, the blood ow in small intestine was signi cantly decreased (P<0.01) when the gavage of 140 mg/kg Dasatinib was administered for 90 minutes, and the blood ow of the small intestine continued to decrease at 135 min (P<0.01). When the mice were given by gavage 210 mg/kg Dasatinib for 45 min, the blood ow in the small intestine was signi cantly decreased (P<0.01), and the blood ow in the small intestine was continuously decreased at 90 and 135 min (P<0.01).

RUS reverted the Intestinal barrier leakage in C57BL/6 subjected to Dasatinib
We found that oral Dasatinib caused a decrease in intestinal blood ow. Therefore, we used the Evans blue method to detect the effects of Dasatinib-induced small intestinal vascular leakage in mice. The results are shown in Fig. 2A. Compared with the control group, the leakage rate of Evans blue in the small intestine of the model group was signi cantly increased (P<0.001), and the inhibitory effect of RUS at the high dose was the best (P<0.001). This indicates that RUS has an obvious effect on intestinal vascular leakage caused by Dasatinib As shown in Fig. 2C, 2D ,RUS at 30μg/kg reversed the blood ow reduction caused by Dasatinib at 90min(P<0.05). HE staining showed that RUS alleviated Dasatinib-induced intestinal barrier destruction and hemorrhage.

RUS ameliorated the expression of junction and adhesion proteins in C57BL/6subjected to Dasatinib
We investigated the effect of RUS on connexin expression by Western blot. The results of protein imprinting showed that compared with the control group, the expression of junction proteins ZO-

RUS reverted the barrier leakage in HUVECs subjected to Dasatinib.
As the Fig.5 shows that the HUVECs were treated with RUS (0.01-1μM), subsequently exposed to 2h of Dasatinib (150 nM), and the barrier protective effect of RUS was evaluated using EB albumin assays in HUVECs.The results demonstrated that treatment with RUS can ameliorates endothelial barrier function after 2 h of Dasatinib(P<0.01).We also observed the effect of RUS on the changes in F-actin using an immuno uorescence assay. The results demonstrated that RUS stabilized the cytoskeleton and reduced the incidence of cell contraction leading to cell shrinkage in HUVECs subjected to Dasatinib . Considering the role of the ROCK/MLC pathway in microvascular endothelial barrier function, we also used western blotting to evaluate the effect of RUS on the expression of proteins associated with the ROCK/MLC pathway. The results showed that treatment with RUS (0.1μM and 1μM) inhibited ROCK1 inhibited, and reduced MLC phosphorylation (P<0.01 & P<0.05). These results indicated that the ROCK/MLC pathway was also involved in the RUS-induced improvement in microvascular endothelial barrier function.

Discussion
The side effects of clinical drugs have always been a major problem for patients' rehabilitation. Drugs that cause serious adverse reactions, even included some commonly used clinical drugs, such as oral antiplatelet drugs, warfarin, oral hypoglycemic drugs, etc [2] .Common adverse reactions include bleeding events, hypoglycemia, arrhythmia, and so on lack effective preventive intervention and monitor methods [30] .Therefore, it is of great signi cance to nd and develop bioactive molecules that can prevent adverse drug reactions.
In order to nd a way to ameliorate the hemorrhagic side effects induced by Dasatinib in clinical practice, we selected Ruscogenin, an active ingredient derived from the natural drug, Ophiopogon japonicus. Experiments have shown that RUS can signi cantly inhibit Dasatinib-induced blood ow decreased at intestinal, through by Evans blue shown that RUS can improved intestinal vascular leakage, and upregulated the expression of connexin ZO-1, Occludin, VEcadherin, and inhibited ROCK activation and MLC phosphorylation.RUS Inhibit the remodeling of the endothelium of blood vessels, exerting the protective effect of the endothelium and inhibiting the adverse reactions caused by Dasatinib.
In order to simulate the clinical use of Dasatinib better [31] , we observed it by high-dose oral administration Dasatinib and found that RUS can display a certain protective effect.In patients with intracerebral hemorrhage, monitoring found that the patient's cerebral blood ow decreased [22] , so we suspected that monitoring intestinal blood ow can re ect intestinal bleeding. The experimental results showed that after administration of Dasatinib by gavage, the intestinal blood ow of mice signi cantly decreased (see Fig. 1).Through this way,we provide a monitoring method for patients who need long-term to take Dasatinib or similar drugs that can cause adverse events like bleeding. After oral administration of Dasatinib, the small intestine structure of the mice was destroyed, bleeding increased, and vascular leakage increased. Pre-administration of RUS reversed the decrease in blood ow and reduced leakage of Evans Blue(see Fig. 2).
Only recently, the intestinal vascular endothelium is recognized as an important barrier in the intestine [32][33] .Vascular endothelial cell is one of the main components of intestinal mucosa.Protecting the intestinal endothelial barrier and maintain vascular stability is become a new way to treat intestinal diseases [34][35][36] . The destruction of connexin will affect the integrity of intestinal blood vessels, increase the permeability of blood vessels, and cause leakage of blood vessels. Therefore, connexin plays a signi cant role in stabilizing intestinal permeability.Tight junctions are essential for regulating intestinal permeability [37][38] . The tight junction proteins in EC include ZO-1, claudin-5, occludin and several JAMs. Previous studies have shown that ZO-1, occludin protein could be regarded as a characteristic marker of TJ, and downregulation of these proteins lead to undermining the tight junctions [39] . Among them, ZO-1 is a junctional adaptor protein that interacts with multiple other junctional components.It can regulate the formation of blood vessels in vitro and in vivo, maintain the connection tension, and at the same time, ZO-1 binds to Factin and is involved in the regulation of cytoskeleton by actomyosin [40] .The endothelial cytoskeleton plays an important role in maintaining the structure of endothelial cells.VE-cadherin is a protein speci cally expressed in endothelial cells and plays an important role in maintaining adhesion junctions.
The steady-state dynamics of VE-cadherin at AJs is a critical determinant of AJ integrity [41] .There is a close relationship between VE-cadherin and ZO-1 [40] . A recent study shows that protecting the endothelial barrier has become a new strategy for treating in ammatory bowel disease [42][43] .All in all, adherens junctions and tight junctions play an important role in maintaining the endothelial barrier. Early administration of RUS can protect the expression of connexin. Compared with the model group, the expressions of ZO-1, VE-cadherin, and Occludin are all increased, and the results are the same in HUVECs in vitro(see Fig. 3 and Fig.6).
The cytoskeletal protein F-actin polymerization produces a mechanical force to change the cell morphology. Therefore, the integrity of the endothelial cytoskeletal protein F-actin structure is the basic structural for maintaining the barrier function of endothelial cells. Dasatinib phosphorylates MLC via the ROCK pathway [24,44] ,induces the polymerization of F-actin structure, remodels the cytoskeleton, and destroys the barrier function of endothelial cells. In view of the function of RUS to protect endothelial cells, we speculate that RUS can inhibit ROCK activation, reduce MLC phosphorylation, reduce cytoskeleton remodeling, restore connexin expression, and play a protective role by interfering with the ROCK-MLC signaling pathway(see Fig.4 and Fig.6).
Dasatinib has excellent e cacy in the clinical treatment of leukemia, but as a protein kinase inhibitor, it is bound to affect the function of the endothelial barrier. It is well known that many different phosphatases and kinases can act as regulators of endothelial barrier function [45][46][47] . After taking Dasatinib, it destroys the original regulatory function, causing damage to the endothelium, which causes numerous adverse reactions. Therefore, before we use these drugs with a high risk of side effects , we can match to some drugs with hemostasis, barrier protection and regulation related signaling pathways to bring more bene ts to patients.Some natural products have good development potential [48] .
Ruscogenin is derived from traditional Chinese medicine, Ophiopogon japonicus,it can anti-in ammatory [49] , anti-thrombosis and protective barrier function [19] . It also can prevent the occurrence of hemorrhagic events.Due to the lack of effective interventions for the bleeding side effects caused by Dasatinib, we considered that Ruscogenin has the function of protecting the endothelial barrier, and there are no clear side effects reported, so we assumed that Ruscogenin can improve the side effects caused by Dasatinib through protects barrier.The use of traditional Chinese medicine to regulate the side effects of other drugs has been initially recognized as a new treatment strategy [50] .

Conclusion
In summary, we provided a new animal model for studying the side effects of Dasatinib, and provided a method for clinical monitoring of gastrointestinal bleeding in patients, and found that a natural product can alleviate the side effects caused by Dasatinib. Whether RUS is effective for long-term use of Dasatinib or gastrointestinal bleeding caused by Dasatinib in pathological conditions, and whether RUS can protect the vascular endothelial barrier through other signaling pathways remains to be explored [51][52][53] . This study broadens the clinical application of RUS in the future, provides pharmacological evidence for RUS prevention and treatment of hemorrhagic side effects, and provides clues for the subsequent development of natural drugs.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by the "Double First-Class"University project(CPU2018GF07), the Fundamental Research Funds for the Central Universities(2632019ZD17).