A mechanism study on the synergistic effects of rifapentine and fluconazole against fluconazole-resistant Candida albicans in vitro.

Candida albicans (C. albicans) is one of the most common clinical isolates of systemic fungal infection. Long-term and inappropriate use of antifungal drugs can cause fungal resistance, which poses a great challenge to the clinical treatment of fungal infections. The combination of antifungal drugs and non-antifungal drugs to overcome the problem of fungal resistance has become a research hotspot in recent years. Our previous study found that the combination of rifapentine (RFT) and fluconazole (FLC) has a significant synergistic against FLC-resistant C. albicans. The present study aimed to further verify the synergistic effect between FLC and RFT against the FLC-resistant C. albicans 100, and explore the underlying mechanism. The growth curve and spot assay test not only showed the synergistic effect of FLC and RFT on FLC-resistant C. albicans in vitro but exhibited a dose-dependent effect on RFT, indicating that RFT may play a principal role in the synergic effect of the two drugs. Flow cytometry showed that the combined use of RFT and FLC arrested cells in the G2/M phase, inhibiting the normal division and proliferation of FLC-resistant C. albicans. Transmission electron microscopy (TEM) demonstrated that FLC at a low concentration could still cause a certain degree of damage to the cell membrane in the FLC-resistant C. albicans, as represented by irregular morphologic changes and some defects observed in the cell membrane. When FLC was used in combination with RFT, the nuclear membrane was dissolved and the nucleus was condensed into a mass. Detection of the intracellular drug concentration of fungi revealed that the intracellular concentration of RFT was 31-195 fold that of RFT alone when it was concomitantly used with FLC. This indicated that FLC could significantly increase the concentration of RFT in cells, which may be due to the damage caused to the fungal cell membrane by FLC. In short, the present study revealed a synergistic mechanism in the combined use of RFT and FLC, which may provide a novel strategy for the clinical treatment of FLC-resistant C. albicans.

San Huang Xiao Yan recipe modulates the HMGB1-mediated abnormal inflammatory microenvironment and ameliorates diabetic foot by activating the AMPK/Nrf2 signalling pathway.

BACKGROUND: Diabetic foot (DF) is one of the serious complications of diabetes and lacks of therapeutic drugs. Abnormal and chronic inflammation promoting foot infection and wound healing delay are the main pathogenesis of DF. The traditional prescription San Huang Xiao Yan Recipe (SHXY) has been used in the clinical treatment of DF for several decades as approved hospital experience prescription and showed remarkable therapeutic effect, but the mechanisms by which SHXY treats DF are still unclear. PURPOSE: Objectives of this study were to investigate SHXY anti-inflammatory effect on DF and explore the molecular mechanism for SHXY. METHODS: We detected the effects of SHXY on DF in C57 mouse and SD rat DF models. Animal blood glucose, weight and wound area were detected every week. Serum inflammatory factors were detected by ELISA. H&E and Masson's trichrome were used to observe tissue pathology. Single-cell sequencing data reanalysis revealed the role of M1 macrophages in DF. Venn analysis showed the co-target genes between DF M1 macrophages and compound-disease network pharmacology. Western blotting was used to explored target protein expression. Meanwhile, RAW264.7 cells were treated with drug-containing serum of SHXY to further unravel the roles of target proteins during high glucose-induced inflammation in vitro. The Nrf2 inhibitor ML385 was used on RAW 264.7 cells to further explore the relationship between Nrf2, AMPK and HMGB1. The main components of SHXY were analysed by HPLC. Finally, the treatment effect of SHXY on DF were detected on rat DF model. RESULTS: In vivo, SHXY can ameliorate inflammatory, accelerate wound healing and upregulate expression of Nrf2, AMPK and downregulate of HMGB1. Bioinformatic analysis showed that M1 macrophages were the main inflammatory cell population in DF. Moreover, the Nrf2 downstream proteins HO-1 and HMGB1 were potential DF therapeutic targets for SHXY. In vitro, we also found that SHXY increased AMPK and Nrf2 protein levels and downregulated HMGB1 expression in RAW264.7 cells. Inhibiting the expression of Nrf2 impaired the inhibition effect of SHXY on HMGB1. SHXY promoted Nrf2 translocation into the nucleus and increased the phosphorylation of Nrf2. SHXY also inhibited HMGB1 extracelluar release under high glucose. In rat DF models, SHXY also exhibited significant anti-inflammatory effect. CONCLUSION: The SHXY activated AMPK/Nrf2 pathway to suppress abnormal inflammation on DF via inhibiting HMGB1 expression. These findings provide novel insight into the mechanisms by which SHXY treats DF.

Development and validation of a sensitive LC-MS/MS method for determination of intracellular concentration of fluconazole in Candida albicans.

Systemic candidiasis is the fourth leading cause of healthcare-associated infections worldwide. The combination therapy based on existing antifungal agents is well-established to overcome drug resistance and restore antifungal efficacy against drug-resistant strains. In this study, a simple and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to quantify the intracellular fluconazole (FLC) content in the opportunistic human fungal pathogen Candida albicans. The cell lysates were prepared by lysing C. albicans cells with Precellys homogenizers and FLC was extracted with methylene chloride. The entire extraction approach was simple, precise and reliable. The extracts were separated on a Zorbax SB-C18 column using a mobile phase of acetonitrile (solvent A) and deionized water plus 0.1% formic acid. FLC and ketoconazole (KCZ, internal standard) were monitored in positive mode using electrospray ionization source. The multiple reaction monitoring transitions (precursor to product) were monitored for FLC m/z 307.1 → 238.2 and for the internal standard KCZ m/z 531.2 → 489.1. The linear for this method were in the range from 5.0 to 1000.0 ng/mL. The precision and accuracy of the samples were relative standard deviations (RSD) < 1.0% for intra-day and RSD < 0.51% for inter-day. The overall recovery of FLC from samples was higher than 77.61%. Furthermore, this method was successfully applied and validated in 36 clinical isolated strains. Taken together, we established a highly accurate, efficient, and reproducible method for quantifying the intracellular content of FLC in C. albicans.

Ruan Jian Qing Mai Recipe Inhibits the Inflammatory Response in Acute Lower Limb Ischemic Mice through the JAK2/STAT3 Pathway.

Ruan jian qing mai recipe (RJQM) is an empirical prescription for treating arteriosclerosis obliterans (ASO). However, the mechanism of RJQM recipe-mediated ASO attenuation has not yet been elucidated. Therefore, this study aimed to explore the mechanism by which the RJQM recipe relieves ASO in a mouse model of lower limb ischemia, which was established by ligating and breaking the femoral artery of the left lower limb. The surgical groups were divided into the ischemic group, beraprost sodium group, low-dose RJQM group, medium-dose RJQM group, and high-dose RJQM group. Normal mice were set as the control group. The blood flow of the lower limb was examined on days 7 and 14. At the end of animal procedures, blood samples were collected, and the rectus femoris of the left lower limb were harvested. Results revealed that mice in the ischemic group demonstrated low blood flow. Additionally, hematoxylin and eosin, and Masson staining results showed that inflammation of the rectus femoris was obvious in the ischemia group, and the level of fibrosis was increased. Blood flow was recovered in all treatment groups compared to the ischemic group, and the inflammatory infiltration and fibrosis of the rectus femoris were relieved after RJQM treatment. The serum levels of interleukin (IL)-17A and IL-21 were decreased, and the expression of JAK2/STAT3 proteins was inhibited in all RJQM treatment groups compared to the ischemia group. Furthermore, the improvement of IL-17A, IL-21, and rectus femoris fibrosis was more obvious with increasing treatment time. In conclusion, RJQM can effectively alleviate ASO and promote the recovery of lower limb blood flow by regulating the JAK2/STAT3 signaling pathway to reduce the inflammatory response.

Effects of oestrogen on vulvovaginal candidosis.

As a frequently occurring infectious disease mainly caused by Candida albicans, vulvovaginal candidosis (VVC) affects more than 100 million women worldwide every year. Multiple factors that influence C. albicans colonisation have been linked to the incidence of VVC, including high levels of circulating oestrogen due to pregnancy, the use of oral contraceptives, and hormone replacement therapy. This review provides an overview of the current understanding of the mechanism(s) by which oestrogen contributes to VVC, which might provide meaningful guidance to the prevention and treatment of this disease.

Design, synthesis, and structure-activity relationship studies of novel fused heterocycles-linked triazoles with good activity and water solubility.

Triazoles with fused-heterocycle nuclei were designed and evaluated for their in vitro activity on the basis of the binding mode of albaconazole using molecular docking, along with SAR of antifungal triazoles. Tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine and tetrahydro-thiazolo[5,4-c]pyridine nuclei were preferable to the other four fused-heterocycle nuclei investigated. Potent in vitro activity, broad spectrum and better water solubility were attained when triazoles containing nitrogen aromatic heterocycles were attached to these two nuclei. The most potent compounds 27aa and 45x, with low hERG inhibition and hepatocyte toxicity, both exhibited excellent activity against Candida, Cryptococcus, and Aspergillus spp., as well as selected fluconazole-resistant strains. A high water-soluble compound 58 (the disulfate salt of 45x) displayed unsatisfactory in vivo activity because of its poor PK profiles. Mice infected with C.alb. SC5314 and C.alb. 103 (fluconazole-resistant strain) and administered with 27aa displayed significantly improved survival rates. 27aa also showed favorable pharmacokinetic (PK) profiles.