Temperature-sensitive hydrogel for rectal perfusion improved the therapeutic effect of Kangfuxin liquid on DSS-induced ulcerative colitis mice: The inflammation alleviation and the colonic mucosal barriers repair

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Abstract

Kangfuxin liquid (KFX) is a Chinese medicine extracted from Periplaneta americana dried worms, which presented the bioactive functions of anti-inflammation and promoting the gastrointestinal mucosal barriers repair. But the low availability of KFX exposed to the distal colon affects its therapeutic effect on ulcerative colitis. Herein, an in situ hydrogel containing KFX was designed by using temperature-sensitive poloxamer 407 (P-407) as material for rectal administration. Three KFX-P formulations with different P407 concentrations (17%, 20% and 25%) were designed and screened by detecting the gelation time, gelation temperature and mechanical strength of hydrogel. P407 in these formulations was able to be completely dissolved in KFX at 4 ℃ and then was in situ gelled at 37 ℃ to form a semisolid hydrogel. Moreover, the gelation time, the gelation temperature and the mechanical strength of KFX-P hydrogel are highly dependent on P407 concentration. With P407 concentration increasing, both the gelation time and gelation temperature of KFX-P accordingly decreased and the gelation temperature range becomes narrowed; while the mechanical strength increased. KFX-P-20% displayed the moderate gelation temperature (28–30 ℃), the short gelation time (26 s) and the moderate mechanical strength (G′ = 4.2 × 103 Pa), which was chosen for animal study. Thereafter, ulcerative colitis mice model (UC) was established by dextran sulfate sodium (DSS) and the therapeutic effect of KFX-P on UC was evaluated by inflammation symptoms relief, colon length, colonic MPO level and colonography. After rectal administration of KFX or KFX-P, the symptoms including diarrhea and hematochezia (DAI scores), weight loss and spleen swelling were significantly hindered. Meanwhile, the colonic MPO level in these groups was significantly decreased in comparison with PBS treatment. But the therapeutic effect of KFX-P was better than KFX. Besides, the morphology and mucosal barrier of colon were evaluated by HE staining, ZO-1 and claudin-5 staining. The mucosa epithelium layer, crypt, muscle layer mucosa and submucosa were also well repaired after KFX-P treatment. The strong fluorescence of ZO-1 and claudin-5 were uniformly distributed along the whole epithelial mucosa after KFX-P treatment, indicating the effective repairing of the colonic mucosal barrier. Collectively, the temperature-sensitive KFX-P for rectal delivery could effectively promote the repair of the colon mucosal barrier and inhibit the colonic inflammation in DSS-induced mice, which may be a potential strategy for UC treatment.

Introduction

Ulcerative colitis (UC), which usually afflicted the proximity of the rectum, is a chorionic inflammatory bowel disorders that characterized by severe intestinal inflammation and the superficial mucosal ulcerations (Neurath, 2019). UC presents the clinical characteristics of recurrent attacks, non-healing, high risk of cancer and poor prognosis, which has been considered as one of the modern refractory diseases by WHO. Continuous or recurrent colitis is a significant cause of colon fibrotic stenosis and colon cancer (Hering and Schulzke, 2009). The causes of UC are believed to be multi-factorial including genetic predisposition, environmental factors, and alterations in the immune system, and disruption in the integrity of the intestinal epithelium and dysbiosis in the gut microbiome. Both of intestinal mucosal barrier disruption and immune dysregulation have been shown to play important roles in intestinal inflammation (Xavier and Podolsky, 2007). Current therapies including 5-ASA, immunosuppressant and antibiotics are mainly focused on treating symptoms. They are often expensive and ineffective in the long term. Moreover, the more stubborn inflammation is easily developed after multiple treatments (Hazel and O'Connor, 2020).

Kangfuxin liquid (KFX) is a Chinese medicine extracted from Periplaneta americana dried worms. Many active components such as polyhydric alcohols, anti-bacterium peptides and mucin are included in KFX, which presented the bioactive functions of acid suppression, anti-inflammation, improving gastrointestinal mucosal microcirculation, promoting granulation tissue hyperplasia and accelerating diseased tissue regeneration. In recent years, a large number of clinical studies have shown that KFX combined with 5-amino salicylic acid has a good effect in the treatment of UC. Moreover, KFX has been approved for the treatment of gastrointestinal ulcer in China (Li et al., 2018, Lu et al., 2019). Pharmacological mechanism of KFX on UC alleviation was associated with inhibiting the levels of NF-κB, IL-1β, TNF-α and INF-γ, and up-regulating the expression of EGF and HGF in colonic mucosa.

KFX was usually orally or rectally administrated as the liquid form for gastrointestinal ulcer in clinic. However, it is difficult for these traditional dosage forms to stay in the colonic mucosa after oral administration, and there are defects such as the loss and discharge of drugs after rectal administration. The poor availability and short retention of KFX in the colon tissue largely compromised its therapeutic effect on ulcerative colitis. Moreover, large volume and multiple administrations regime are usually required for rectal dosage forms to exert the effective therapeutic response, which cause secondary colonic mucosa damage and reduce the sensitivity of the intestine. The poor availability and short retention of KFX in the colon tissue largely compromised its therapeutic effect on ulcerative colitis.

Poloxamer is a polyoxyethylene-polyoxypropylene-polyoxyethylene typed block copolymer, which has the low toxicity and the excellent compatibility with other chemicals. Poloxamer 407(P407), one of the commercially available polymers, contains 70% of polyoxyethylene units and 30% of polyoxypropylene blocks. P407 at concentrations of 17% or higher in aqueous solution exhibits the unique property of reversible thermal gelation. These preparations transform from low-viscosity solutions to semisolid gels upon heating from 4℃ to body temperature (37 ℃) (Xia et al., 2019). Such gels can be localized near the administration site and wide spread discharge of the drug can be minimized. These properties make P407 an attractive vehicle for controlled drug delivery in many pharmaceutical products (Galvis et al., 2019).

In this study, P407 as a thermo-sensitive material was directly dissolved in KFX liquid to construct a rectally administrated KFX-P hydrogel for UC alleviation. KFX-P hydrogel was expected to be gelled in situ in the colon lumen, extend the retention time of KFX and thus improve its therapeutic effect on UC. The concentration of P-407 in formulation was firstly screened by according to the parameters such as the gelling time, the gelling temperature and the mechanical strength of hydrogel. The retention of KFX-P hydrogel in colon mucosa was also investigated by in vivo imaging after rectal administration. Finally, the therapeutic effect of KFX-P hydrogel on ameliorating colitis symptoms was evaluated on DSS-induced colitis mice. The inflammatory remission and recovery of the intestinal epithelial barrier were confirmed by detecting inflammatory cytokines and tight junction proteins.

Section snippets

Materials

Dextran sodium sulfate (DSS) was purchased from Sigma-Aldrich; Poloxamer 407 (BASF) was obtained from BASF; Kangfuxin (KFX) liquid was purchased from Chashan drug store (Wenzhou, China); ZO-1 Rabbit pAb (A0659, ABclonal®), CLDN5 Rabbit pAb (A10207, ABclonal®), and myeloperoxidase kit were purchased from AB clonal (Wuhan, China).

Preparation of temperature-sensitive KFX-P hydrogel

An amount of P407 were dispersed in 100 mL KFX liquid under continuous stirring, followed by placement in refrigerator at 4 ℃ for overnight to form a light yellow

Preparation and characterization of temperature-sensitive KFX hydrogel

Poloxamer not only possess the hydrophilic PEO segment but also contains the hydrophobic PPO segment. The hydrophilic segment PEO forms an intermolecular hydrogen bond with water, making poloxamer soluble in cold water. The intermolecular hydrogen bond is easily broke as temperature increases, which resulted in the dehydration of PEO-PPO-PEO in the extended state and the formation of hydrogel (Xu et al., 2017). Poloxamer 407(P407), one of the commercially available polymers, contains 70% of PEO

Discussions

Ulcerative colitis is characterized by the mucosal inflammation and ulceration. The afflicted colon is not self-healing in clinic because of the repeated attacks of colonic ulceration. At present, small molecule anti-inflammatory drugs or antibiotics are still the main means to relieve the inflammatory reaction for patient with ulcerative colitis. But the outcome for these treatments was limited and the recurrence of inflammation was completely not prevented. Instead of inhibiting inflammatory

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was supported by Zhejiang Provincial Natural Science Foundation (Grant No. LY20H300002) and Wenzhou municipal science and technology bureau (Grant No. Y20180183).

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    The first two authors contributed equally to this work.

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