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BACKGROUND: Plasma exosomal microRNAs (miRNAs) have been used as potential biomarkers for various diseases and have been investigated for their possible involvement in the pathogenesis of vitiligo. However, the miRNA expression profile of plasma exosomes in patients with non-segmental vitiligo (NSV) has not been determined yet. OBJECTIVE: To screen differentially expressed microRNAs in plasma exosomes derived from patients with NSV and explore their roles in the pathogenesis of NSV. METHODS: High-throughput sequencing was performed to determine the expression profiles of exosomal miRNAs in NSV. The effect of upregulated miR-1469 in NSV circulating exosomes on natural killer (NK) cells was further investigated using various molecular biological techniques. RESULTS: MiR-1469 was identified as a candidate biomarker whose expression was significantly increased in circulating exosomes of NSV patients. Circulating exosomes were internalized by NK cells and increased NK cell proliferation viability and IFN-γ secretion capacity delivering miR-1469. Further studies revealed that the upregulation of CD122, the predicted target of miR-1469, could partially reverse the effect of miR-1469 on natural killer cells. CONCLUSION: Alterations in plasma exosomal cargo occur in NSV and appear to contribute to NK cell dysfunction. Exosomal miR-1469 may be a biomarker of disease activity and could be used as a therapeutic drug target against innate immunity in NSV patients. The present study provides new insights into the role of exosomal miRNAs in NSV and suggests a novel miR-1469-CD122-IFN-γ pathway of NK cell underlying pathogenesis of NSV.

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Vitiligo is a common autoimmune skin disease caused by autoreactive CD8+ T cells. The diverse effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on immune cell metabolism and proliferation have made it an interesting candidate as a supporting therapeutic option in various autoimmune diseases. This study aimed to elucidate the immunomodulatory effects of 1,25(OH)2D3 in vitiligo. Cross-sectional relationships between serum 1,25(OH)2D3 levels and disease characteristics were investigated in 327 patients with vitiligo. The immunomodulatory and therapeutic effects of 1,25(OH)2D3 were then investigated in vivo and in vitro, respectively. We found that 1,25(OH)2D3 deficiency was associated with hyperactivity of CD8+ T cells in the vitiligo cohort. In addition, 1,25(OH)2D3 suppressed glycolysis by activating the AMP-activated protein kinase (AMPK) signaling pathway, thereby inhibiting the proliferation, cytotoxicity and aberrant activation of CD8+ T cells. Finally, the in vivo administration of 1,25(OH)2D3 to melanocyte-associated vitiligo (MAV) mice reduced the infiltration and function of CD8+ T cells and promoted repigmentation. In conclusion, 1,25(OH)2D3 may serve as an essential biomarker of the progression and severity of vitiligo. The modulation of autoreactive CD8+ T cell function and glycolysis by 1,25(OH)2D3 may be a novel approach for treating vitiligo.

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Tripterygium glycoside tablet (TGT) has been used clinically to alleviate diabetic nephropathy (DN) for decades. However, the mechanism of its anti-DN has not been fully clarified. The aim of this study was to elucidate molecular mechanism of TGT in repairing renal function injury. The results of biochemical parameters and renal histopathology implied that TGT intervention could attenuate creatinine, albumin excretion rate and histological injury of kidney in DN mouse model. Moreover, UHPLC-QTOF-MS/MS-based untargeted metabolomic analysis indicated that 11 metabolites in kidney of mice with DN were restored after TGT treatment, and the most prominent metabolic alteration was triglyceride (TG) metabolism. Mechanistically, TGT effectively improved the function of impaired kidney by promoting TG catabolism via modulation of adipose triglyceride lipase in DN mice. Our findings identified the link between circulating metabolites and DN, suggesting that it might be a possibility to intervene in DN by targeting metabolism.

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Circulating exosomal microRNAs have been used as potential biomarkers for various disorders. However, to date, the microRNA expression profile of circulating exosomes in patients with segmental vitiligo (SV) has not been identified. Thus, we aimed to identify the expression profile of circulating exosomal microRNAs and investigate their role in the pathogenesis of SV. Our study identified the expression profile of circulating exosomal microRNAs in SV and selected miR-493-3p as a candidate biomarker whose expression is significantly increased in circulating exosomes and perilesions in patients with SV. Circulating exosomes were internalized by human primary keratinocytes and increased dopamine secretion in vitro. Furthermore, miR-493-3p overexpression in keratinocytes increased dopamine concentration in the culture supernatant, which led to a significant increase in ROS and melanocyte apoptosis as well as a decrease in melanocyte proliferation and melanin synthesis in the coculture system by targeting HNRNPU. We also confirmed that HNRNPU could bind to and regulate COMT, a major degradative enzyme of dopamine. Hence, circulating exosomal miR-493-3p is a biomarker for SV, and the miR-493-3p/HNRNPU/COMT/dopamine axis may contribute to melanocyte dysregulation in the pathogenesis of SV.

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BACKGROUND: Melanogenesis is a multistep process in which melanocytes produce melanin pigments within melanosomes. However, the roles played by the biological factors and pathways in this process are not yet fully understood. OBJECTIVE: To investigate the role of ATP-binding cassette subfamily B member 6 (ABCB6) in the regulation of melanogenesis in vitro. METHODS: Real-time PCR and western blotting were used to assess the knockdown efficiency of ABCB6 in MNT-1 and PIG1 stable cell lines. Cleavage by NaOH was used to determine melanin content, while the number of melanosomes was examined for each stage by transmission electron microscopy. Immunofluorescence microscopy was used to evaluate endogenous protein location. Differentially expressed genes were detected using RNA sequencing, and gene expression was assessed by quantitative real-time PCR. KEGG mapping was used for pathway enrichment analysis. Co-immunoprecipitation was used for protein-protein interactions analysis. RESULTS: We found that ABCB6 inhibition could impair melanocyte maturation and melanin production in human melanoma (MNT-1) and immortalized human melanocyte (PIG1) cell lines. Moreover, ABCB6 knockdown inhibited the protein expression of melanocyte inducing microphthalmia-associated transcription factor (MITF) and its three downstream melanogenic enzymes (TYR, TYRP1 and TYRP2). Mechanistically, we revealed that ABCB6 could interact with and modulate glycogen synthase kinase 3 beta (GSK3-ß) to exert its biological effect on melanogenesis. CONCLUSION: Our findings suggest that ABCB6 is a key regulator of melanogenesis via the GSK3-ß/ß-catenin signaling pathway. However, further in-depth studies are essential to uncover the relationship between ABCB6 and pigmentation disorders.

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PURPOSE: Oxidative stress is considered a major determinant in the pathogenesis of vitiligo. Methylcobalamin (MeCbl) is an activated form of vitamin B12 that regulates inflammatory factors, counters oxidative stress, and reduces apoptosis in many disease models. However, the specific mechanism of MeCbl repigmentation against vitiligo is unknown. In this study, we explored the effect of MeCbl on melanocytes following hydrogen peroxide (H2O2)-induced oxidative stress. METHODS: We established an oxidative stress model using the immortalized human normal melanocyte cell line PIG1. We used a Cell Counting Kit-8 (CCK-8) to detect drug cytotoxicity, and we measured the melanin content of cells using the NaOH method. Intracellular oxidative damage was assessed by flow cytometry and antioxidant enzyme detection kits. In addition, we assessed the presence of apoptosis by flow cytometry and Western blots. We explored the underlying mechanisms of MeCbl during oxidative stress in melanocytes by analyzing the results of experiments based on real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and laser scanning confocal immunofluorescence microscopy. Finally, we repeated the experiments after applying an inhibitor to block the Nrf2 pathway. RESULTS: We found that MeCbl treatment enhanced cell viability, increased melanin content, reduced intracellular reactive oxygen species (ROS) accumulation, increased the activities of antioxidant enzyme superoxide dismutase (SOD) and catalase (CAT), reduced melanocyte apoptosis, and up-regulated the expression of the Nrf2/HO-1 pathway. Moreover, the protective effects of MeCbl were significantly weakened after inhibiting the Nrf2/HO-1 pathway. CONCLUSION: Our results indicate that MeCbl attenuated the H2O2-induced oxidative stress in melanocytes by activating the Nrf2/HO-1 pathway, this suggests that MeCbl may be an effective treatment against vitiligo.

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Hyperlipidemia can easily cause atherosclerosis and induce cardiovascular and cerebrovascular diseases. Red yeast rice (RYR) contains a variety of active ingredients and is commonly used as medicine and food, and has pharmacological effects such as lowering blood lipids. In this study, we select Monascus strain SHM1105 with a high yield of Monacolin K and monascus pigment (PIG), and studied the effects of the RYR and PIG fermented by this strain on blood lipids, intestinal flora, and liver transcriptome in hyperlipidemia model rats. The experimental results show that, compared with the high-fat model group, the weight growth rate, liver weight ratio, kidney weight ratio, spleen weight ratio, and fat weight ratio of rats in the gavage lovastatin (LOV), RYR, and PIG group were all significantly decreased (p < 0.05). Intervention with RYR and PIG can significantly reduce the serum TC, TG, and LDL-C levels, which has the effect of lowering blood lipids. The 16SrDNA sequencing results showed that the ratio of Firmicutes/Bacteroidetes decreased significantly (p ≤ 0.01) after the intervention of LOV, RYR, and PIG; the abundance of the ratio of Lachnospiraceae, Ruminococcaceae, Prevotellaceae, and Bacteroidales-S24-7-group also changed. The combined analysis of transcriptome and metabolome showed that lovastatin, RYR, and PIG can all improve lipid metabolism in rats by regulating Steroid hormone biosynthesis, Glycerolipid metabolism, and the Arachidonic acid metabolism pathway. In addition, RYR and PIG also have a unique way of regulating blood lipids. Although a lot of research on the lipid-lowering components of Monascus rice and the single pigment component of Monascus has been carried out, the actual application is RYR and pigments as mixtures, as a mixture of RYR and PIG contains a variety of biologically active ingredients, and each component may have a synergistic effect. Hence it has a lipid-lowering mechanism that lovastatin does not have. Therefore, RYR and PIG are effective in reducing lipid potential development and can be utilized in functional foods.

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OBJECTIVE: Renal fibrosis generally results in renal failure during the end stage of chronic renal diseases. There are many cell factors including E-cadherin, α-SMA, and TGF-ß1 influencing deposition of extracellular matrix and leading to renal fibrosis. As the most important and widely-used therapy for various diseases in China for thousands of years, traditional Chinese medicine (TCM) provides a novel treatment for renal fibrosis. For clinical application, we explore the effect of Bu-Shen-Huo-Xue formula (BSHX), a traditional Chinese herbal formula, on E-cadherin and α-SMA in rats with 5/6 nephrectomy. MATERIALS AND METHODS: Sprague-Dawley rats were subjected to 5/6 nephrectomy to induce chronic renal failure (CRF); they were divided into three groups including a CRF control group, a BSHX group, and a Cozaar group, and compared with a normal control group. After 8 weeks of therapy with the respective drug, E-cadherin, α-SMA, and TGF were detected by immunohistochemistry assays in renal tissues. RESULTS: As the immunohistochemistry assays indicated, BSHX could significantly enhance the expression of E-cadherin and depress the levels of α-SMA and TGF-ß1 expression in rats' renal tissues with 5/6 nephrectomy. CONCLUSION: BSHX can effectively relieve the renal fibrosis in rats with 5/6 nephrectomy via the change of cell factor levels including enhancement of the expression of E-cadherin and depression of the levels of α-SMA and TGF-ß1 expression.
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Renal ischemia reperfusion injury (RIRI) refers to the irreversible damage for renal function when blood perfusion is recovered after ischemia for an extended period, which is common in clinical surgeries and has been regarded as a major risk for acute renal failures (ARF) that is accompanied with unimaginably high morbidity and mortality. Hypoxia during ischemia followed by reoxygenation via reperfusion serves as a major event contributing to cell apoptosis, which has been widely accepted as the vital pathogenesis in RIRI. Preventing apoptosis in renal tubular epithelial cell has been considered as effective method for blocking RIRI. In this paper, we established a hypoxia/reoxygenation (H/R) injury model in human proximal tubular epithelial HK-2 cells. Here, we found increased SPHK1 levels in H/R injured HK-2 cells, which could be significantly down regulated after berberine treatment. Berberine has been reported to exert a protective effect on H/R-induced apoptosis of HK-2 cells. So, in our present study, we planned to investigate whether SPHK1 participated in the anti-apoptosis process of berberine in H/R injured HK-2 cells. Our study confirmed the protective effect of berberine against H/R-induced apoptosis in HK-2 cells through promoting cells viability, inhibiting cells apoptosis, and down-regulating p-P38, caspase-3, caspase-9 as well as SPHK1, while up regulating the ratio of Bcl-2/Bax. However, SPHK1 overexpression in HK-2 cells induced severe apoptosis, which can be significantly ameliorated with additional berberine treatment. We concluded that berberine could remarkably prevent H/R-induced apoptosis in HK-2 cells through down-regulating SPHK1 expression levels, and the mechanisms included the suppression of p38 MAPK activation and mitochondrial stress pathways.

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A specific and sensitive LC-MS/MS method was established for the simultaneous determination of bergenin, protocatechuic acid and gallic acid, the main active constituents of Saxifraga stolonifera (L.) Meerb. herb, in rat plasma. After fully validated, the method was applied to the comparative pharmacokinetic studies of the three compounds orally administered alone and in combination in the S. stolonifera extract, respectively. The results showed that the pharmacokinetic parameters, including Cmax, Tmax, AUC, CLz/F, MRT0-∞, were significantly different for both bergenin and protocatechuic acid in the extract as compared to the corresponding compounds administered alone. However, the pharmacokinetic behavior of gallic acid in the extract did not differ from that administered alone. Further studies found that quercetin, coexisting in the herb extract, significantly decreased the glucuronidation of bergenin through inhibiting the activities of UGT1A1 and UGT1A3, and reduced the metabolism of protocatechuic acid by inhibiting the activity of catechol-O-methyltransferase. Quercetin and other flavonoids occurring in the S. stolonifera extract might increase the absorption and improve the bioavailability of bergenin and protocatechuic acid by slowing down the liver metabolism. The findings provide a good guidance for the development and clinical application of S. stolonifera.

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In this study, a rapid and highly sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was established for the quantification of paeoniflorin in rat plasma. The analyte and the internal standard (IS), hyperoside, were extracted from rat plasma via liquid-liquid extraction with ethyl acetate. LC separation was carried on a Phenomenex Luna C18 column within 2.5 min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization and operated in the multiple reaction monitoring and negative ion modes. The precursor to product ion transitions monitored for paeoniflorin and the IS were m/z 524.8→449.0 and 463.1→300.0, respectively. The assay was validated with a linear range of 0.02~25.6 µg/mL for paeoniflorin. The intra- and inter-day precisions (relative standard deviation%) were within 9.7%, and the recoveries were greater than 65.2%. Paeoniflorin was proven to be stable during all sample storage, preparation and analytical procedures. The method was successfully applied to a pharmacokinetic study of the Baixiangdan Capsule in eight female rats.

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BACKGROUND: Xuesaitong dispersible tablet (XST) product has been clinically proven to be effective for treating cardio-cerebrovascular disease. Furthermore, herb-drug interactions between the XST product and drugs that are commonly co-administered, such as aspirin (ASA), must be explored to ensure safe clinical use. STUDY DESIGN AND METHODS: The current study aims to investigate whether the XST product interacts with ASA when they are administered concomitantly to ensure safety and efficacy. A ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of ginsenoside Rg1 (Rg1), ginsenoside Rd (Rd), notoginsenoside R1 (R1) and salicylic acid (SA) in rat plasma to investigate the pharmacokinetic interaction of XST and ASA in blood stasis model rats. RESULTS AND CONCLUSION: The ASA and XST combination noticeably altered R1 and Rg1 absorption, distribution and disposition. This study indicates that co-administration of XST and ASA can cause an apparent herb-drug pharmacokinetic interaction in blood stasis model rats.

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FmEG from Fomitiporia mediterranea is a non-modular endoglucanase composed of a 24-amino acids extension and 13-amino acids linker-like peptide at the N-terminus and a 312-amino acids GH5 catalytic domain (CD) at the C-terminus. In this study, six FmEG derivatives with deletion of N-terminal fragments or fusion with an extra family 1 carbohydrate-binding module (CBM1) was constructed in order to evaluate the contribution of CBM1 to FmEG processivity and catalytic activity. FmEG showed a weak processivity and released cellobiose (G2) and cellotriose (G3) as main end products, and cellotriose (G4) as minor end product from filter paper (FP), but more amount of G4 was released from regenerated amorphous cellulose (RAC). All derivatives had similar activity on carboxymethylcellulose (CMC) with the same optimal pH (7.0) and temperature (50°C). However, fusing an extra CBM1 to FmEG△24 or FmEG△37 with flexible peptide significantly improved its processivity and catalytic activity to FP and RAC. Overall, 1.79- and 1.84-fold increases in the soluble/insoluble product ratio on FP, and 1.38- and 1.39-fold increases on RAC, compared to FmEG△24, were recorded for CBM1-FmEG△24 and CBM1-linker-FmEG△24, respectively. Meanwhile, they displayed 2.64- and 2.67-fold more activity on RAC, and 1.68- and 1.77-fold on FP, respectively. Similar improvement was also obtained for CBM1-linker-FmEG△37 as compared with FmEG△37. Interestingly, fusion of an extra CBM1 with FmEG also caused an alteration of cleavage pattern on insoluble celluloses. Our results suggest that such improvements in processivity and catalytic activity may arise from CBM1 binding affinity. The N-terminal 24- or 37-amino acids may serve as linker for sufficient spatial separation of the two domains required for processivity and catalytic activity. In addition, deletion of the N-terminal 24- or 37-amino acids led to significant reduction in thermostability but not the enzymatic activity.

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Bergenin, isolated from the herb of Saxifrage stolonifera Curt. (Hu-Er-Cao) has hepatoprotective, anti-inflammatory, antitussive, and neuroprotective activities. The aim of the present study was to establish a simple, rapid, and sensitive RP-HPLC method for determination of bergenin in rat plasma and compare its oral pharmacokinetic behaviors in normal and CCl4-induced hepatic injury rats. With norisoboldine as an internal standard, chromatographic separation was performed on a C18 analytical column with acetonitrile and water (11 : 89, V/V) containing 0.1% formic acid as the mobile phase. A good linearity was obtained over the range of 100-10 000 ng·mL-1. The lower limit of quantification was 50 ng·mL-1. The developed method was successfully applied to a study of the pharmacokinetic difference of bergenin (100 mg·kg-1) between normal and hepatic injury rats after oral administration. Marked alterations of pharmacokinetic parameters in hepatic injury rats were observed. Compared to normal rats, the AUC(0-∞) of bergenin in hepatic injury rats was elevated to 2.11-fold and Cmax was increased by 130%, whereas CL value was only 55% of the normal rats, suggesting that the systemic exposure of bergenin was significantly increased under hepatic injury status.

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A novel mixer was developed to improve the performance of flat-plate photobioreactors (PBRs). The effects of mixer were theoretically evaluated using computational fluid dynamics (CFD) according to radial velocity of fluid and light/dark cycles within reactors. The structure parameters, including the riser width, top clearance, clearance between the baffles and walls, and number of the chambers were further optimized. The microalgae culture test aiming at validating the simulated results was conducted indoor. The results showed the maximum biomass concentrations in the optimized and archetype reactors were 32.8% (0.89 g L(-1)) and 19.4% (0.80 g L(-1)) higher than that in the control reactor (0.67 g L(-1)). Therefore, the novel mixer can significantly increase the fluid velocity along the light attenuation and light/dark cycles, thus further increased the maximum biomass concentration. The PBRs with novel mixers are greatly applicable for high-efficiency cultivation of microalgae.

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Medium screening and optimization is one of the most important preconditions for photoautotrophic cultivation of microalgae. Although, it has been widely conducted indoors, little work performed outdoors. There are enormous differences between indoor and outdoor conditions, especially for light intensity, temperature and their diurnal or annual fluctuations, which would greatly influence microalgae growth. No data shows whether the differences would lead to different results on medium screening and optimization. In present study, medium screening for the photoautotrophic cultivation of Chlorella pyrenoidosa was carried out indoors and outdoors firstly, and then the selected medium was optimized. The results showed that F-Si medium is the optimum both under indoor and outdoor conditions. Based on F-Si medium, nutrients were optimized as follows: NaNO3 500mgl(-1), NaH2PO4·2H2O 7.7mgl(-1) and FeCl3·6H2O 6.30mgl(-1). With the optimized medium, the biomass, lipid content and productivity were all significantly higher both indoors and outdoors.

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As the optimal source of astaxanthin, Haematococcus pluvialis was cultured for commercial production of astaxanthin through two continuous phases: cell growth and astaxanthin induction. In this study, the efficiency of an attached system for producing astaxanthin from H. pluvialis was investigated and compared to that of the suspended system (bubble column bioreactor) under various conditions. Results showed that this attached system is more suitable for photoinduction of H. pluvialis than the suspended bioreactor. Under the optimal conditions, the astaxanthin productivity of the attached system was 65.8 mg m(-2)d(-1) and 2.4-fold of that in the suspended system. This attached approach also offers other advantages over suspended systems, such as, producing astaxanthin under a wide range of light intensities and temperatures, saving water, ease to harvest cells, resisting contamination. Therefore, the attached approach can be considered an economical, environmentally friendly and highly-efficient technology for producing astaxanthin from H. pluvialis.

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Photoautotrophic cultivation with heterotrophic cells as seeds (heterotrophic cells/photoautotrophic cultivation) is an effective way for the development of microalgal biofuel, but its development potential from the point of process optimization has not been investigated in literatures. To evaluate this, the optimizations of medium and culture conditions for Chlorella ellipsoidea were studied. In the heterotrophic stage, the biomass concentration reached 11.04 g/L with the optimized medium in flask, which were 28.0% higher than that with the original medium, and the biomass concentration reached 73.89 g/L in 5-L fermenter. In the photoautotrophic stage, the culture medium and conditions were studied in a 2-L column photobioreactor. The maximum biomass concentration, lipid content and lipid productivity reached 1.62 g/L, 36.34% and 6.1 mg/(L·h) under the optimal photoautotrophic conditions. The lipids were mainly composed of C16-C18 fatty acids, which were raw material suitable for biodiesel. After optimization, heterotrophic cells/photoautotrophic cultivation can significantly improve the capacity of biofuel production by Chlorella ellipsoidea, this method is also expected to be an efficient way for the cultivation of other microalgae that can grow heterotrophically.

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Yijiejing (Doctor's Mirror), the first novel focused on medical ethics in the late Qing dynasty, aimed at meliorating medical ethical atmosphere by criticizing disorders among medical practitioners. Some contents of this novel were the same as those in Yijie Xianxingji (Revelation of Medical Community). By comparison between the two books and investigation on Doctor's Mirror's introduction, preface and advertisement in the newspaper at that time, we could find that the author of Doctor's Mirror was not the novelist LU Shi-e, but YU Wen-yao, the author of Yijie Xianxingji. Distribution of Yijie Xianxingji was stopped soon after its publication by the Commercial Press for its allusion to a famous doctor in Shanghai. Two years later, YU made some slight modifications the details of the novel's characters and his book was published with a different title Doctor's Mirror and the pen-name 'medical hermit among scholars'.