Enzyme/ROS dual-sensitive nanoplatform with on-demand Celastrol release capacity for enhanced ulcerative colitis therapy by ROS scavenging, microbiota rebalancing, inflammation alleviating.

BACKGROUND: The oral administration of drugs for treating ulcerative colitis (UC) is hindered by several factors, including inadequate gastrointestinal stability, insufficient accumulation in colonic lesions, and uncontrolled drug release. METHODS: A multiple sensitive nano-delivery system comprising ß-cyclodextrin (CD) and 4-(hydroxymethyl)phenylboronic acid (PAPE) with enzyme/reactive oxygen species (ROS) sensitivity was developed to load celastrol (Cel) as a comprehensive treatment for UC. RESULTS: Owing to the positive charge in the site of inflamed colonic mucosa, the negatively charged nanomedicine (Cel/NPs) could efficiently accumulate. Expectedly, Cel/NPs showed excellent localization ability to colon in vitro and in vivo tests. The elevated concentration of ROS and intestinal enzymes in the colon microenvironment quickly break the CD, resulting in Cel release partially to rebalance microbiota and recover the intestinal barrier. The accompanying cellular internalization of residual Cel/NPs, along with the high concentration of cellular ROS to trigger Cel burst release, could decrease the expression of inflammatory cytokines, inhibit colonic cell apoptosis, promote the macrophage polarization, scavenge ROS, and regulate the TLR4/NF-κB signaling pathway, which certified that Cel/NPs possessed a notably anti-UC therapy outcome. CONCLUSIONS: We provide a promising strategy for addressing UC symptoms via an enzyme/ROS-sensitive oral platform capable of releasing drugs on demand.

Exploring the neuroprotection of the combination of astragaloside A, chlorogenic acid and scutellarin in treating chronic cerebral ischemia via network analysis and experimental validation.

Chronic cerebral ischemia (CCI) primarily causes cognitive dysfunction and other neurological impairments, yet there remains a lack of ideal therapeutic medications. The preparation combination of Astragalus membranaceus (Fisch.) Bunge and Erigeron breviscapus (Vant.) Hand.-Mazz have been utilized to ameliorate neurological dysfunction following cerebral ischemia, but material basis of its synergy remains unclear. The principal active ingredients and their optimal proportions in this combination have been identified through the oxygen and glucose deprivation (OGD) cell model, including astragaloside A, chlorogenic acid and scutellarin (ACS), and its efficacy in enhancing the survival of OGD PC12 cells surpasses that of the combination preparation. Nevertheless, mechanism of ACS against CCI remains elusive. In this study, 63 potential targets of ACS against CCI injury were obtained by network pharmacology, among which AKT1, CASP3 and TNF are the core targets. Subsequent analysis utilizing KEGG and GO suggested that PI3K/AKT pathway may play a crucial role for ACS in ameliorating CCI injury. Then, a right unilateral common carotid artery occlusion (rUCCAO) mouse model and an OGD PC12 cell model were established to replicate the pathological processes of CCI in vivo and in vitro. These models were utilized to explore the anti-CCI effects of ACS and its regulatory mechanisms, particularly focusing on PI3K/AKT pathway. The results showed that ACS facilitated the restoration of cerebral blood flow in CCI mice, enhanced the function of the central cholinergic nervous system, protected against ischemic nerve cell and mitochondrial damage, and improved cognitive function and other neurological impairments. Additionally, ACS upregulated the expression of p-PI3K, p-AKT, p-GSK3ß and Bcl-2, and diminished the expression of Cyto-c, cleaved Caspase-3 and Bax significantly. However, the PI3K inhibitor (LY294002) partially reversed the downregulation of Bax, Cyto-c and cleaved Caspase-3 expression as well as the upregulation of p-AKT/AKT, p-GSK3ß/GSK3ß, and Bcl-2/Bax ratios. These findings suggest that ACS against neuronal damage in cerebral ischemia may be closely related to the activation of PI3K/AKT pathway. These results declared first time ACS may become an ideal candidate drug against CCI due to its neuroprotective effects, which are mediated by the activated PI3K/AKT pathway mitigates mitochondrial damage and prevents cell apoptosis.

Tumor Microenvironment ROS/pH Cascade-Responsive Supramolecular Nanoplatform with ROS Regeneration Property for Enhanced Hepatocellular Carcinoma Therapy.

The low targeted drug delivery efficiency, including poor tumor accumulation and penetration and uncontrolled drug release, leads to the failure of cancer therapy. Herein, a multifunctional supramolecular nanoplatform loading triptolide (TPL/PBAETK@GA NPs) was fabricated via the host-guest interaction between glycyrrhetinic-acid-modified poly(ethylene glycol)-adamantanecarboxylic acid moiety and reactive oxygen species (ROS)/pH cascade-responsive copolymer poly(ß-amino esters)-thioketal (TK)-ß-cyclodextrin. TPL/PBAETK@GA NPs could accumulate in hepatocellular carcinoma (HCC) tissue effectively, mediated by nanoscale advantage and GA' recognition to specific receptors. The elevated concentration of ROS in tumor microenvironment (TME) quickly breaks the TK linkages, causing the detachment of shell (cyclodextrin) CD layer. Then, the accompanying negative-to-positive charge-reversal of NPs was realized via the PBAE moiety protonation under the slightly acidic TME, significantly enhancing the NPs' cellular internalization. Remarkably, the pH-responsive endo/lysosome escape of PBAE core triggered intracellular TPL burst release, promoting the cancer cell apoptosis, autophagy, and intracellular ROS generation, leading to the self-amplification of ROS in TME. Afterward, the ROS positive-feedback loop was generated to further promote size-shrinkage and charge-reversal of NPs. Both in vitro and in vivo tests verified that TPL/PBAETK@GA NPs produced a satisfactory anti-HCC therapy outcome. Collectively, this study offers a potential appealing paradigm to enhance TPL-based HCC therapy outcomes via multifunctionalized supramolecular nanodrugs.

Study on the mechanism of natural polysaccharides on the deastringent effect of Triphala extract.

This present study investigated the masking effect of high methoxyl pectin, xanthan gum, and gum Arabic on the astringency of the traditional herbal formula Triphala and further examined the mechanism of polysaccharide reducing astringency. Results of sensory evaluation and electronic tongue illustrated that 0.6 % pectin, 0.3 % xanthan gum, and 2 % gum Arabic had a substantial deastringent effect. The polyphenols in Triphala are basically hydrolysable tannins, which with high degree of gallic acylation may be the main astringent component of Triphala. Moreover, the three polysaccharides can combine with ß-casein through CO and NH groups to form soluble binary complexes and decrease the secondary structure of ß-casein. When polysaccharides were added to the Triphala-protein system, polyphenol-protein precipitation was also diminished, and they were capable of forming soluble ternary complexes. Consequently, the competition between polysaccharides and polyphenols for binding salivary proteins and the formation of ternary complexes help decrease the astringency of Triphala.

The material basis of astringency and the deastringent effect of polysaccharides: A review.

Astringency is a feeling of dryness in the mouth. Microscopically, it is manifested in the diversity of ingredients and mechanisms that can cause astringency, astringent components are mainly flavonoids, phenolic acids, tannin and other polyphenols components. Macroscopically, it is manifested in the rich variety of foods with astringent taste, because polyphenols are common secondary metabolites of plants. With the improvement of people's living standards, the demand for reducing or removing astringency in food and medicine is also increasing, and polysaccharides, as commonly used flavoring agents and food additives, have become the ideal choice for decreasing astringency. In this paper, the material basis, molecular mechanism, possible pathways and related cases of polysaccharides moderating of astringency are mainly reviewed, so as to illustrate the feasibility of polysaccharides decreasing of astringency and provide a reference for reducing the astringency of food and drugs.

Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells.

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in the efficacy and mechanism between the preparation combination and the component combination in order to explore the two herb combination strategy to treat injured PC12 cells. Cobalt chloride (CoCl2) combined with a glucose-free medium was employed to induce oxidative damage of PC12 cells. Then, the optimal combination of Astragalus mongholicus (Ast) and Erigeron breviscapus (Eri) injection was selected and combined following uniform design methods after screening their safe and effective concentration on PC12 cells. Further, the component combination screened comprises 10 µM astragaloside A, 40 µM scutellarin, and 75 µM chlorogenic acid in two herbs. Then, MTT, Annexin V-FITC/PI, immunofluorescence, and Western blot analysis were used to evaluate the efficacy and mechanism of the preparation combination and the component combination on injured PC12 cells. The results showed that the optimal preparation combination for cell pro-survival was Ast injection and Eri capsule with a concentration of 6:1.8 (µM). The component combination (10 µM astragaloside A, 40 µM scutellarin, and 75 µM chlorogenic acid) was more effective than the preparation combination. Both combinations remarkably reduced apoptotic rate, the fluorescence intensity of caspase-3, and intracellular reactive oxygen species (ROS) level; meanwhile, they upregulated the expression levels of p-Akt/Akt, Bcl-2/Bax, and Nrf2. These effects were more evident in the component combination. In conclusion, both combinations can inhibit the injury induced by CoCl2 combined with a glucose-free medium on PC12 cells, thus promoting cell survival. However, the efficiency of the component combination over the preparation combination may be due to its stronger regulation of the PI3K/Akt/Nrf2 signaling pathway related to oxidative stress and apoptosis.

[Connotation and mitigation of polyphenolic astringency in Chinese medicine].

Taste is one of the important factors in the design of oral drug preparations. Polyphenols are the secondary metabolites produced in the growth process of Chinese medicine with a variety of physiological activities. However, astringency perceived from polyphenols tastes uncomfortable. As one of the true taste of Chinese medicine, astringency with drying, rough, and wrinkled sensation, seriously affects the texture of Chinese medicine and the compliance of patients. Due to the universality of polyphenolic astringency in Chinese medicine and the weakness of modern research, this study systematically reviewed and summarized the latest research on the mechanism of polyphenolic astringency, the astringency evaluation method, and the astringency-mitigation technology. Through comprehensively analyzing the quantification methods, such as sensory evaluation, animal preference evaluation, chemical evaluation, bionic evaluation, and polyphenol-protein interaction evaluation, the direction of overall astringency assessment with "unified dimension" was proposed. Since the characteristics of Chinese medicine and the mechanism of polyphenolic astringency did not reach a consensus, this study proposed the idea of astringency mitigation suitable for Chinese medicine. This study is intended to deepen the understanding of astringency associated with Chinese medicine, and establish a real and objective astringency evaluation method for Chinese medicine, thus promoting the technique of astringency mitigation of polyphenolic Chinese medicine preparations from trial and error to science.

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis.

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a universally used traditional Chinese medicine) as an example. Ten batches of genuine Chuanmutong varieties and five batches of related adulterants were analyzed and compared based on the high-performance liquid chromatography (HPLC) fingerprints combined with chemometrics, including cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least-squares discrimination analysis (OPLS-DA). In addition, the content of ß-sitosterol was determined. The control chemical fingerprint of Chuanmutong was established, and 12 common peaks were identified. The similarity between the fingerprint of 10 batches of genuine Chuanmutong varieties and the control fingerprint was 0.910-0.989, while the similarity of five batches of adulterants was only 0.133-0.720. Based on the common peaks in the chromatogram, 15 batches of samples were classified into three content levels by PCA, and were aggregated into four categories by CA, achieving a clear distinction between authentic Chuanmutong and adulterants of Chuanmutong. Further, seven differential components that can effectively identify authentic Chuanmutong and adulterants of Chuanmutong were found through OPLS-DA. The ß-sitosterol content of 10 batches of genuine Chuanmutong varieties was 97.53-161.56 µg/g, while the ß-sitosterol content of the five batches of adulterants varied greatly, among which the ß-sitosterol content of Clematis peterae Hand.-Mazz. and Clematis gouriana Roxb. Var. finetii Rehd. et Wils. was significantly lower than that of authentic varieties of Chuanmutong. The HPLC index component content and chemical fingerprint multi-pattern recognition method established in this study provide a new strategy for effectively identifying authentic Chinese medicinal materials and related adulterants.

Optimization and Evaluation of the Thermosensitive In Situ and Adhesive Gel for Rectal Delivery of Budesonide.

Budesonide is a glucocorticoid for the treatment of ulcerative colitis (UC). The current study aims to develop a thermosensitive in situ and adhesive gel for rectal delivery of budesonide. HPMC K4M was selected as the adhesive agent based on the adhesive force and the effect on gel performance. The formulation of gel was optimized by using the central composite design-response surface methodology (CCD-RSM); a mathematical model was successfully developed to predict desired formulations as well as to analyze relationships between the amount of Pluronic F-127, Pluronic F-68, and HPMC K4M and the performances of gel. Based on CCD-RSM, a thermosensitive in situ and adhesive gel consisting of 0.002% budesonide, 0.74% HPMC, 4.87% F-68, and 19.0% F-127 was developed. Furthermore, the in vivo behavior of gel was evaluated in Sprague-Dawley rats. In comparison with budesonide solution, rectal administration of budesonide gel at 0.1 mg/kg in rats showed relative bioavailability of 230% with significant increase in rectum uptake.

Targeting Delivery of Rapamycin with Anti-Collagen IV Peptide Conjugated Fe3O4@Nanogels System for Vascular Restenosis Therapy.

Coronary arterial disease (CAD) remains the leading cause of death globally. Although percutaneous coronary interventions (PCI) are most of the important clinical procedure for CAD treating, unfortunately, vascular restenosis is proved as the major drawback of PCI. Because of the complex nature of the restenotic process, the efficacy of drug administration is emphasized. Targeting drug delivery systems become a promising experimental approach for restenosis therapy. Hence, we design and fabricate a thermo/pH-responsive nanogel system with the magnetic inner core as the multifunctional nanocarrier for drug delivery and MRI/fluorescence imaging. To this end, NIPAm-based Fe3O4 core-shell structure nanogel is synthesized as the responsive nanosystem for rapamycin (RAPA) delivery, and the surface conjugation with anti-collagen IV peptide makes the nanosystem an ideal candidate for targeting delivery of RAPA. Based on its stimuliresponsive properties, the nanosystem shows desirable intracellular release behavior of RAPA and significantly reduces the adverse effect of RAPA. The in vitro cytotoxicity evaluations present the biosafety profiles and antiproliferation performance of the drug-loaded nanogels. Meanwhile, the magnetic Fe3O4 inner core shows enhanced T2 weight sensitivity, providing a high potential for imaging-guiding therapy. In the balloon angioplasty model, targeting nanogels are demonstrated accumulation at the injured site of artery. Compared with the non-targeting nanogels, treatment with peptide conjugated nanogels attenuates neointimal hyperplasia more effectively. The biochemical assays further reveal that the enhanced restenosis prevention effect is contributed to the selective release of RAPA at the injured sites of artery, which notably potentiate the nanosystem as a systemically targeting delivered treatment.

Inducing Controlled Release and Increased Tumor-Targeted Delivery of Chlorambucil via Albumin/Liposome Hybrid Nanoparticles.

Liposomes possess good biocompatibility and excellent tumor-targeting capacity. However, the rapid premature release of lipophilic drugs from the lipid bilayer of liposomes has negative effect on the tumor-targeted drug delivery of liposomes. In this study, a lipophilic antitumor drug-chlorambucil (CHL)-was encapsulated into the aqueous interior of liposomes with the aid of albumin to obtain the CHL-loaded liposomes/albumin hybrid nanoparticles (CHL-Hybrids). The in vitro accumulative release rate of CHL from CHL-Hybrids was less than 50% within 48 h, while the accumulative CHL release was more than 80% for CHL-loaded liposomes (CHL-Lip). After intravenous injection into rats, the half-life (t 1/2ß = 5.68 h) and maximum blood concentration (C max = 4.58 µg/mL) of CHL-Hybrids were respectively 1.1 times and 3.5 times higher than that of CHL-Lip. In addition, CHL-Hybrids had better tumor-targeting capacity for it significantly increased the drug accumulation in B16F10 tumors, which contributed to the significantly control of tumor growth compared with CHL-Lip. Furthermore, CHL-Hybrid-treated B16F10 melanoma-bearing mice displayed the longest median survival time of 30.0 days among all the treated groups. Our results illustrated that the proposed hybrids drug delivery system would be a promising strategy to maintain the controlled release of lipophilic antitumor drugs from liposomes and simultaneously facilitate the tumor-targeted drug delivery.

Casitas B-Lineage Lymphoma RING Domain Inhibitors Protect Mice against High-Fat Diet-Induced Obesity and Insulin Resistance.

The casitas b-lineage lymphoma (c-Cbl) is an important adaptor protein with an intrinsic E3 ubiquitin ligase activity that interacts with E2 proteins such as UbCH7. c-Cbl plays a vital role in regulating receptor tyrosine kinase signaling. c-Cbl involves in whole-body energy homeostasis, which makes it a potential target for the treatment of type 2 diabetes and obesity. In the present study, we have designed two parental peptides and 55 modified peptides based on the structure of UbCH7 loop L1 and L2. Thirteen of the modified peptides showed increased inhibitory activity in a fluorescence polarization-based assay. In the in vivo proof of study principle, mice treated with peptides 10, 34, 49 and 51 were protected against high-fat diet-induced obesity and insulin resistant. These inhibitors may potentially lead to new therapeutic alternatives for obesity and type 2 diabetes.

Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators.

The E3 ubiquitin protein ligase Casitas B-lineage Lymphoma (Cbl) proteins and their binding partners play an important role in regulating signal transduction pathways. It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding specific small-molecule regulators of PPIs remains a significant challenge due to the fact that the interfaces involved in PPIs are not well suited for effective small molecule binding. We report the development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule regulators of Cbl (RING) domain. The FP assay was used to measure binding affinities and inhibition constants of UbCH7 peptides and small molecule regulators of Cbl (RING) domains, respectively. In order to rule out promiscuous, aggregation-based inhibition, two assay conditions were developed and compared side by side. Under optimized conditions, we screened a 10,000 natural compound library in detergent-free and detergent-present (0.01% Triton X-100) systems. The results indicate that the detergent-present system is more suitable for high-throughput screens. Three potential compounds, methylprotodioscin, leonuride and catalpol, have been identified that bind to Cbl (RING) domain and interfere with the Cbl (RING)-UbCH7 protein-protein interaction.

[Preparation of kangfuxin colon targeting micro-pellets].

OBJECTIVE: To prepare coated micro-pellets of pH-dependent and enzyme-dependent kangfuxin colon targeting delivery system, to make them go to colon, then release, educe partial effect. METHOD: We eploy pan-pill to prepare simple pellets, and prepare tunicatus pellets with fluidized bed coating. We investigated the preparation and parameter of pellets, so, we bolting the best shaping and tunicatus artwork. RESULT: The ingredients for preparing the micro-pellets are 125% starch +2% CMC-Na, and add 30% ethanol to be binder, pellets were coated with Eudragit S100 to prepare ph-dependent and pectin-HPMC to prepare enzyme-dependent colon targeting micro-pellets. CONCLUSION: We get two micro-pellets of pH-dependent and enzyme-dependent kangfuxin colon targeting.

[In vivo/in vitro evaluation of Kangfuxin colon targeting capsules].

OBJECTIVE: To evaluate the release in fixed position of pH-dependent and enzyme-dependent Kangfuxin colon targeting capsules in vivo and in vitro. METHOD: The dissolution was tested in vitro and X-ray radiography was used for the evaluation in vivo. RESULT: After two hours pH-dependent colon targeting in man-made colon fluid, medicine release in fixed position on the whole, colon loc-release. Add enzyme into man-made colon, when enzyme-dependent colon targeting in it, then medicine release quickly, mainly release in fixed position; The conveying time in vivo of pH-dependent and enzyme-dependent capsules have big individuality difference. In the experiment, disintegration is stabilize among individuales, between 2.0-3.5 hours. CONCLUSION: Kangfuxin colon targeting capsules of two principles all release in fixed position to achieve the goal.

[Preparation and in vivo evaluation of pH and time dependent Yuchangning tablets for colon-specific delivery].

OBJECTIVE: Taking Chinese compound medicine Yuchangning as a research model, prepared the pH and time dependent Yuchangning tablets for colon-specific delivery (PT-YT-CSD), and evaluated the releasing property in vitro. METHOD: The coating prescription is filtered by the release extent of matrine and oxymatrine in vitro and the wicking rate of the tablet, which including the category and proportion of film forming agent and porogen, the sort and dosage of fluidizing agent, the increment of weight after coating and so on. The releasing property of the preparation is evaluated by the dissolution tests in vitro through measuring the content of matrine and oxymatrine content. RESULT: The preparation method of the PYTCSD: After prepared plain tablets, the 95% alcoholic solution of EC and Eudragit II are mixed in a 7:3 EC: Eudragit II ratio and then added in DEP up to 10% of the coating amount, reduced the alcohol concentration to 4% by diluting with ethonal. Tablet was coated by the alcohol solution and the weight of the plain tablet was increased by 3%. The dissolution tests in vitro indicated that matrine and oxymatrine were not dissolved in the simulated gastric juice after 2 h. The accumulative quantities of matrine and oxymatrine were less than 10% in the simulated intestinal juice after 4 h. The quantities of matrine and oxymatrine are 75.7% and 76.8% in the simulated colon juice after 1 h. CONCLUSION: The PYTCSD was prepared and the preparation could fulfil the aim of delivering in the specific colon in vitro.