MP-13, a novel chimeric peptide of morphiceptin and pepcan-9, produces potent antinociception with limited side effects.

Pharmacological investigations have substantiated the potential of bifunctional opioid/cannabinoid agonists in delivering potent analgesia while minimizing adverse reactions. Peptide modulators of cannabinoid receptors, known as pepcans, have been investigated before. In this study, we designed a series of chimeric peptides based on pepcans and morphiceptin (YPFP-NH2). Here, we combined injections of pepcans and morphiceptin to investigate the combination treatment of opioids and cannabis and compared the analgesic effect with chimeric compounds. Subsequently, we employed computational docking to screen the compounds against opioid and cannabinoid receptors, along with an acute pain model, to identify the most promising peptide. Among these peptides, MP-13, a morphiceptin and pepcan-9 (PVNFKLLSH) construct, exhibited superior supraspinal analgesic efficacy in the tail-flick test, with an ED50 value at 1.43 nmol/mouse, outperforming its parent peptides and other chimeric analogs. Additionally, MP-13 displayed potent analgesic activity mediated by mu-opioid receptor (MOR), delta-opioid receptor (DOR), and cannabinoid type 1 (CB1) receptor pathways. Furthermore, MP-13 did not induce psychological dependence and gastrointestinal motility inhibition at the effective analgesic doses, and it maintained non-tolerance-forming antinociception throughout a 7-day treatment regimen, with an unaltered count of microglial cells in the periaqueductal gray region, supporting this observation. Moreover, intracerebroventricular administration of MP-13 demonstrated dose-dependent antinociception in murine models of neuropathic, inflammatory, and visceral pain. Our findings provide promising insights for the development of opioid/cannabinoid peptide agonists, addressing a crucial gap in the field and holding significant potential for future research and development. PERSPECTIVE: This article offers insights into the combination treatment of pepcans with morphiceptin. Among the chimeric peptides, MP-13 exhibited potent analgesic effects in a series of preclinical pain models with a favorable side-effect profile.

The intestinal microbiota and metabolic profiles of Strauchbufo raddei underwent adaptive changes during hibernation.

The intestinal microbiota help regulate hibernation in vertebrates. However, it needs to be established how hibernation modulates the gut microbiome and intestinal metabolism. In the present study, we used an artificial hibernation model to examine the responses of the gut microbiota of the Strauchbufo raddei to the environmental changes associated with this behavior. Hibernation significantly lowered the diversity of the microbiota and altered the microbial community of the gut. Proteobacteria, Firmicutes, and Bacteroidota were the major bacterial phyla in the intestines of S. raddei. However, Firmicutes and Proteobacteria predominated in the gut of active and hibernating S. raddei, respectively. Certain bacterial genera such as Pseudomonas, Vibrio, Ralstonia, and Rhodococcus could serve as biomarkers distinguishing hibernating and non-hibernating S. raddei. The gut microbiota was more resistant to environmental stress in hibernating than active S. raddei. Moreover, metabolomics revealed that metabolites implicated in fatty acid biosynthesis were highly upregulated in the intestines of hibernating S. raddei. The metabolites that were enriched during hibernation enabled S. raddei to adapt to the low temperatures and the lack of exogenous food that are characteristic of hibernation. A correlation analysis of the intestinal microbiota and their metabolites revealed that the gut microbiota might participate in the metabolic regulation of hibernating S. raddei. The present study clarified the modifications that occur in the intestinal bacteria and their symbiotic relationship with their host during hibernation. These findings are indicative of the adaptive changes in the metabolism of amphibians under different environmental conditions.

Dynamic changes in and time sequence of ultraviolet B-induced apoptosis in rat corneal epithelial cells / Alterações dinâmicas na sequência temporal da apoptose induzida por radiação ultravioleta B em células epiteliais da córnea de ratos

ABSTRACT Purpose: To systematically examine the dynamic changes and time sequence in corneal epithelial cell apoptosis after excessive ultraviolet B irradiation. Methods: Ultraviolet B (144 mJ/cm2) was used to irradiate rat corneal epithelial cells for 2 h. Cell morphology was observed on differential interference contrast microscopy, and the numbers of the different kinds of apoptotic cells were counted using the ImageJ software. Cell viability was measured with the 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide method. Cell apoptotic rate and loss of mitochondrial membrane potential were detected using flow cytometric analyses. The expression levels of 3 apoptotic genes were measured with real-time quantitative polymerase chain reaction at different time points within 0-24 h after irradiation. Results: After 144-mJ/cm2 ultraviolet B irradiation for 2 h, the expression levels of caspase-8 and Bax were highest at 0 h; furthermore, the mitochondrial membrane potential decreased at 0 h and remained constant for 6 h in a subsequent culture. At 6 h, caspase-3 was activated. The decrease in cell viability and increase in apoptotic rate peaked at 6 h. The caspase-3 expression level decreased within 12-24 h, which led to a decline in apoptotic rate and change in apoptotic stage. Conclusions: The corneal epithelial cells exhibited rapid apoptosis after ultraviolet B irradiation, which was associated with both extrinsic and intrinsic pathways.

RESUMO Objetivos: Explorar sistematicamente as mudanças dinâmicas e a sequência temporal no processo de apoptose de células epiteliais corneanas após excesso de irradiação com ultravioleta B. Métodos: A radiação ultravioleta B (144 mJ/cm2) foi utilizada para irradiar células epiteliais da córnea de rato durante 2h. A morfologia celular foi observada por meio de microscópio de contraste de interferência diferencial, e os números de diferentes tipos de células apoptóticas foram contados e registrados pelo software ImageJ. A viabilidade celular foi medida pelo método brometo de 3- (4, 5-dimetil-2-tiazolil) -2, 5-difenil-2-H-tetrazólio. A taxa apoptótica celular e a perda do potencial da membrana mitocondrial foram detectadas por meio de análises citométricas de fluxo. Os níveis de expressão de três genes apoptóticos foram medidos por reação em cadeia da polimerase quantitativa em tempo real em diferentes momentos dentro de 0-24 h após a irradiação. Resultados: Após 144 mJ/cm2 de irradiação com ultravioleta B por 2h, os níveis de expressão de caspase-8 e Bax foram maiores em 0h; o potencial da membrana mitocondrial diminuiu a 0h e permaneceu constante por 6h na cultura subsequente. Às 6h, a caspase-3 foi ativada. A diminuição da viabilidade celular e o aumento da taxa apoptótica atingiu o pico em 6h. A expressão de caspase-3 diminuiu dentro de 12 - 24 h, levando a um declínio na taxa apoptótica e alteração no estágio apoptótico. Conclusões: As células epiteliais da córnea apresentaram uma apoptose rápida após excesso de irradiação com ultravioleta B, e esse processo foi associado tanto à via extrínseca como à via intrínseca.

Dynamic changes in and time sequence of ultraviolet B-induced apoptosis in rat corneal epithelial cells.

PURPOSE: To systematically examine the dynamic changes and time sequence in corneal epithelial cell apoptosis after excessive ultraviolet B irradiation. METHODS: Ultraviolet B (144 mJ/cm2) was used to irradiate rat corneal epithelial cells for 2 h. Cell morphology was observed on differential interference contrast microscopy, and the numbers of the different kinds of apoptotic cells were counted using the ImageJ software. Cell viability was measured with the 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide method. Cell apoptotic rate and loss of mitochondrial membrane potential were detected using flow cytometric analyses. The expression levels of 3 apoptotic genes were measured with real-time quantitative polymerase chain reaction at different time points within 0-24 h after irradiation. RESULTS: After 144-mJ/cm2 ultraviolet B irradiation for 2 h, the expression levels of caspase-8 and Bax were highest at 0 h; furthermore, the mitochondrial membrane potential decreased at 0 h and remained constant for 6 h in a subsequent culture. At 6 h, caspase-3 was activated. The decrease in cell viability and increase in apoptotic rate peaked at 6 h. The caspase-3 expression level decreased within 12-24 h, which led to a decline in apoptotic rate and change in apoptotic stage. CONCLUSIONS: The corneal epithelial cells exhibited rapid apoptosis after ultraviolet B irradiation, which was associated with both extrinsic and intrinsic pathways.

Acute oral colchicine caused gastric mucosal injury and disturbance of associated microbiota in mice.

Colchicine (COL), an ancient and well-known drug, has been used in clinical practice for centuries. On the other hand, COL has also attracted extensive concerns for its potent toxic effects, especially gastrointestinal adverse reactions (nausea, vomiting, and diarrhea) before clinical symptoms relief. In this study, we used a rodent model to study the effects of COL on gastric mucosa and associated microbiota. The mice were exposed to various concentrations of COL (0.1, 0.5, and 2.5 mg kg-1 body weight per day) for 7 days, and the results showed that COL treatment caused severe gastric mucosal damage, accompanied by a significant decrease in gastric mucosal proinflammatory cytokines (IL-1ß, IL-6, and TNF-α). The 16S rRNA gene sequencing revealed that COL significantly perturbed the gastric microbiota composition and reduced the gastric microbiota diversity in mice. Also, we identified bacterial biomarkers associated with diarrhea, including phylum Firmicutes, class Bacilli, order Lactobacillales, family Lactobacillaceae, genu Lactobacillus, and genu Blautia, suggesting that COL-triggered adverse reactions are closely related to gastric microbial perturbations. Our findings open new paths for understanding the mechanism of COL-related adverse gastrointestinal reactions, broadening the scientific view on the interaction between drugs and host gastrointestinal microbiota.

Protective effect of rapamycin in models of retinal degeneration.

Age-related macular degeneration (AMD) is a complex retinal disease with no viable treatment strategy. The causative mechanistic pathway for this disease is not yet clear. Therefore, it is highly warranted to screen effective drugs to treat AMD. Rapamycin are known to inhibit inflammation and has been widely used in the clinic as an immunosuppressant. This study aimed to investigate the protective effect of rapamycin on the AMD retinal degeneration model. The AMD models were established by injection of 35 mg/kg sodium iodate (NaIO3) into the tail vein. Then the treated mice intraperitoneally received rapamycin (2 mg/kg) once a day. The histomorphological analysis showed that rapamycin could inhibit retinal structure damage and apoptosis. Experiments revealed that rapamycin significantly attenuated inflammatory response and oxidative stress. Our experimental results demonstrated that rapamycin has protected the retinal against degeneration induced by NaIO3. The therapeutic effect was more significant after 7 days of treatment. Therefore, our study potentially provides a powerful experimental support for the treatment of AMD.

Rapamycin ameliorates corneal injury after alkali burn through methylation modification in mouse TSC1 and mTOR genes.

Alkali burn to the cornea is one of the most intractable injuries to the eye due to the opacity resulting from neovascularization (NV) and fibrosis. Numerous studies have focused on studying the effect of drugs on alkali-induced corneal injury in mouse, but fewer on the involvement of alkali-induced DNA methylation and the PI3K/AKT/mTOR signaling pathway in the mechanism of alkali-induced corneal injury. Thus, the aim of this study was to determine the involvement of DNA methyltransferase 3 B-madiated DNA methylation and PI3K/AKT/mTOR signaling modulation in the mechanism of alkali-induced corneal injury in a mouse model. To this end, we used bisulfite sequencing polymerase chain reaction and Western blot analysis, to study the effects of 5-aza-2'-deoxycytidine and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, which inhibit methyltransferase and PI3K respectively, on DNA methylation and expression of downstream effectors of PI3K related to corneal NV, including TSC1 and mTOR genes. The results showed that, after an intraperitoneal injection of rapamycin (2 mg/kg/day) for seven days, the alkali-induced opacity and NV were remarkably decreased mainly by suppressing the infiltration of immune cells into injured corneas, angiogenesis, VEGF expression and myofibroblasts differentiation; as well as by promoting corneal cell proliferation and PI3K/AKT/mTOR signaling. More significantly, these findings showed that epigenetic regulatory mechanisms by DNA methylation played a key role in corneal NV, including in corneal alkali burn-induced methylation modification and rapamycin-induced DNA demethylation which involved the regulation of the PI3K/AKT/mTOR signaling pathway at the protein level. The precise findings of morphological improvement and regulatory mechanisms are helpful to guide the use of rapamycin in the treatment of corneal angiogenesis induced by alkaline-burn.

Colchicine increases intestinal permeability, suppresses inflammatory responses, and alters gut microbiota in mice.

Although colchicine (COL) has been used to treat gout for more than a thousand years, it has been shrouded in a dark history for a long time due to its high toxicity, especially for the gastrointestinal tract. With the widespread clinical application of COL, COL's toxicity to the gastrointestinal tract has raised concerns. This study's objective was to address the exact intestinal toxicity of COL, with particular attention to the effects of COL on gut microbiota homeostasis. The mice were exposed to various dosages of COL (0.1, 0.5, and 2.5 mg kg-1 body weight per day) for a week, and the results showed that COL exposure caused serious intestinal injuries, reducing the relative expression levels of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and tight junction proteins (zo-1, claudin-1, and occludin) in the ileum and colon tissue. The 16S rRNA gene sequencing analysis of mice feces samples revealed that the composition and diversity of intestinal microbiome underwent a profound remodeling at the dosage of 2.5 mg kg-1 body weight per day, which may increase the toxic load in the gut. In addition, elevated levels of diamine oxidase (DAO) and lipopolysaccharide (LPS) in serum indicated that COL increased intestinal permeability, impairing intestinal barrier. In conclusion, our results demonstrate that COL's toxicity to the gut microbiome is compatible with intestinal injuries, inflammatory pathway inhibition, and increased intestinal permeability; our results also represent a novel insight to uncover the adverse reactions of COL in the gastrointestinal tract.