Detection of Tyrosinase Activity and Inhibitor Validation Based on N-GQDs Fluorescence Sensor.

Tyrosinase inhibitors have the ability to resist melanin formation and can be used for clinical and cosmetic, so it is becoming extremely crucial to search a rapid and effective method for detecting t the activity of tyrosinase. In this study, a sensing probe based on Nitrogen-doped graphene quantum dots (N-GQDs) were prepared with carbamide and citric acid. Tyrosinase can oxidize dopamine to dopamine quinone, which can quench the fluorescence of N-GQDs based on the principle of fluorescence resonance energy transfer (FRET) process, and then the detection of tyrosinase activity can be achieved. The result demonstrated that the fluorescence intensity of N-GQDs was a linear correlation with the activity of tyrosinase. Wide detection linear ranges between 0.05 and 5 U/mL and high selectivity. The detection range of tyrosinase was 0.05 to 5 U/mL and LOD of 0.005 U/mL. According to the above, the fluorescence method established in this work could be successfully used for the trace analysis of tyrosinase and it was verified that KA is an inhibitor of tyrosinase.

CEBPA Restrains the Malignant Progression of Breast Cancer by Prompting the Transcription of SOCS2.

The suppressor of cytokine signaling 2 (SOCS2) has been identified to act as a tumor suppressor in breast cancer (BC) progression. However, the action of SOCS2 in macrophage polarization in BC cells has not been reported yet. The qRT-PCR and western blotting were adopted for detecting the levels of mRNAs and proteins. The macrophage M2 polarization was analyzed by flow cytometry. Analyses of cell oncogenic phenotypes and tumor growth were conducted using 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, scratch, Transwell, tube formation assays in vitro, and tumor xenograft assay in vivo, respectively. The interaction between CEBPA (CCAAT Enhancer Binding Protein Alpha) and SOCS2 was confirmed using bioinformatics analysis and dual-luciferase reporter assay. SOCS2 was lowly expressed in BC tissues and cells. Functionally, overexpression of SOCS2 inhibited macrophage M2 polarization, and impaired BC cell proliferation, angiogenesis, and metastasis. Mechanistically, CEBPA bound to the promoter region of SOCS2, and promoted its transcription. A low CEBPA expression was observed in BC tissues and cells. Forced expression of CEBPA also suppressed macrophage M2 polarization, BC cell proliferation, angiogenesis, and metastasis. Moreover, the anticancer effects mediated by CEBPA were abolished by SOCS2 knockdown. In addition, CEBPA overexpression impeded BC growth in nude mice by regulating SOCS2. CEBPA suppressed macrophage M2 polarization, BC cell proliferation, angiogenesis, and metastasis by promoting SOCS2 transcription in a targeted manner.

Fermentation-mediated variations in structure and biological activity of polysaccharides from Tetrastigma hemsleyanum Diels et Gilg.

Variations in the structure and activities of polysaccharides from Tetrastigma hemsleyanum Diels et Gilg fermented by Sanghuangporus sanghuang fungi were investigated. Compare with the unfermented polysaccharide (THDP2), the major monosaccharide composition and molecular weight of polysaccharide after fermentation (F-THDP2) altered dramatically, which caused galactose-induced conversion from glucose and one-third of molecular weight. F-THDP2 had a molecular weight of 1.23 × 104 Da. Moreover, the glycosidic linkage of F-THDP2 varied significantly, a 1, 2-linked α-d-Galp and 1, 2-linked α-d-Manp backbone was established in F-THDP2, which differed from that of 1, 4-linked α-d-Glcp and 1, 4-linked ß-d-Galp in THDP2. In addition, F-THDP2 showed a more flexible chain conformation than that of THDP2 in aqueous solution. Strikingly, F-THDP2 exhibited superior inhibitory effects on HeLa cells via Fas/FasL-mediated Caspase-3 signaling pathways than that of the original polysaccharide. These variations in both structure and biological activities indicated that fermentation-mediated modification by Sanghuangporus sanghuang might a promising novel method for the effective conversion of starch and other polysaccharides from Tetrastigma hemsleyanum Diels et Gilg into highly bioactive biomacromolecules, which could be developed as a potential technology for use in the food industry.

A Highly Water-Soluble Aggregation-Induced Emission Luminogen with Anion-π+ Interactions for Targeted NIR Imaging of Cancer Cells and Type†I Photodynamic Therapy.

The low oxygen dependence of type I photosensitizers (PSs) has made them a popular choice for treating solid tumors. However, the drawbacks of poor water solubility, short emission wavelength, poor stability, and inability to distinguish cancer cells from normal cells limit the application of most type I PSs in clinical therapy. Thereby, developing novel type I PSs for overcoming these problems is an urgent but challenging task. Herein, by utilizing the distinctive structural characteristics of anion-π+ interactions, a highly water-soluble type I PS (DPBC-Br) with aggregation-induced emission (AIE) characteristic and near-infrared (NIR) emission is fabricated for the first time. DPBC-Br displays remarkable water solubility (7.3 mM) and outstanding photobleaching resistance, enabling efficient and precise differentiation between tumor cells and normal cells in a wash-free and long-term tracking manner via NIR-I imaging. Additionally, the superior type I reactive oxygen species (ROS) produced by DPBC-Br provide both specific killing of cancer cells in vitro and inhibition of tumor growth in vivo, with negligible systemic toxicity. This study rationally constructs a highly water-soluble type I PS, which has higher reliability and controllability compared with conventional nanoparticle formulating procedures, offering great potential for clinical cancer treatment.

Plasma exosome proteomics reveals the pathogenesis mechanism of post-stroke cognitive impairment.

Exploration and utilization of exosome biomarkers and their related functions provide the possibility for the diagnosis and treatment of post-stroke cognitive impairment (PSCI). To identify the new diagnostic and prognostic biomarkers of plasma exosome were uzed label-free quantitative proteomics and biological information analysis in PSCI patients. Behavioral assessments were performed, including the Mini-Mental Status Examination (MMSE), the Montreal Cognitive Assessment (MoCA), the Barthel index, the Morse Fall Seale (MFS) between control group (n = 10) and PSCI group (n = 10). The blood samples were collected to analyse the biomarker and differentially expressed proteins of plasma exosome using label-free quantitative proteomics and biological information. The exosomes marker proteins were determined by Western blot. The exosome morphology was observed by transmission electron microscopy. The scores of MMSE and MoCA were significantly decreased in the PSCI group. The PT% and high-density lipoprotein decreased and the INR ratio increased in PSCI group. The mean size of exosome was approximately 71.6 nm and the concentration was approximately 6.8E+7 particles/mL. Exosome proteomics identified 259 differentially expressed proteins. The mechanisms of cognitive impairment are related to regulate the degradation of ubiquitinated proteins, calcium dependent protein binding, cell adhesive protein binding, formation of fibrin clot, lipid metabolism and ATP-dependent degradation of ubiquitinated proteins in plasma exosome of PSCI patients. Plasma levels of YWHAZ and BAIAP2 were significantly increased while that of IGHD, ABCB6 and HSPD1 were significantly decreased in PSCI patients. These proteins might be target-related proteins and provide global insights into pathogenesis mechanisms of PSCI at plasma exosome proteins level.

Aptamer-based nanointerferometer enables amplification-free ultrasensitive detection and differentiation of SARS-CoV-2 variants.

The state-of-the-art SARS-CoV-2 detection methods include qRT-PCR and antibody-based lateral flow assay (LFA) point-of-care tests. Despite the high sensitivity and selectivity, qRT-PCR is slow, expensive and needs well-trained operators. On the other extreme, LFA suffers from low sensitivity albeit its fast detection speed, low detection cost and ease of use. Therefore, the continuing COVID-19 pandemic calls for a SARS-CoV-2 detection method that is rapid, convenient and cost-effective without compromise in sensitivity. Here we provide a proof-of-principle demonstration of an optimized aptamer-based nanointerferometer that enables rapid and amplification-free detection of SARS-CoV-2 spike protein-coated pseudovirus directly from human saliva with the limit of detection (LOD) of about 400 copies per mL. This LOD is on par with that of qRT-PCR, making it 1000 to 100,000-fold more sensitive than commercial LFA tests. Using various combinations of negative selections during the screens for the aptamer targeting the receptor binding domain of the spike protein of SARS-CoV-2, we isolated two aptamers that can distinguish the Omicron and Delta variants. Integrating these two aptamers with LFA strips or the nanointerferometer sensors allows both detection and differentiation of the Omicron and Delta variants which has the potential to realize rapid triage of patients infected different SARS-CoV-2 variants.

Fluorination Enhances NIR-II Emission and Photothermal Conversion Efficiency of Phototheranostic Agents for Imaging-Guided Cancer Therapy.

Phototheranostics with second near-infrared (NIR-II) imaging and photothermal effect have become a burgeoning biotechnology for tumor diagnosis and precise treatment. As important parameters of phototheranostic agents (PTAs), fluorescence quantum yield (QY) and photothermal conversion efficiency (PCE) are usually considered as a pair of contradictions that is difficult to be simultaneously enhanced. Herein, a fluorination strategy for designing A-D-A type PTAs with synchronously improved QY and PCE is proposed. Experimental results show that the molar extinction coefficient (ε), NIR-II QY, and PCE of all fluorinated PTAs nanoparticles (NPs) are definitely improved compared with the chlorinated counterparts. Theoretical calculation results demonstrate that fluorination can maximize the electrostatic potential difference by virtue of the high electronegativity of fluorine, which may increase intra/intermolecular D-A interactions, tighten molecule packing, and further promote the increase of ε, ultimately leading to simultaneously enhanced QY and PCE. In these PTA NPs, FY6-NPs display NIR-II emission extended to 1400 nm with the highest NIR-II QY (4.2%) and PCE (80%). These features make FY6-NPs perform well in high-resolution imaging of vasculature and NIR-II imaging-guided photothermal therapy (PTT) of tumors. This study develops a valuable guideline for constructing NIR-II organic PTAs with high performance.

Preclinical development of a long-acting trivalent bispecific nanobody targeting IL-5 for the treatment of eosinophilic asthma.

BACKGROUND: Eosinophilic asthma is a common subtype of severe asthma with high morbidity and mortality. The cytokine IL-5 has been shown to be a key driver of the development and progression of disease. Although approved monoclonal antibodies (mAbs) targeting IL-5/IL-5R have shown good safety and efficacy, some patients have inadequate responses and frequent dosing results in medication nonadherence. RESULTS: We constructed a novel trivalent bispecific nanobody (Nb) consisting of 3 VHHs that bind to 2 different epitopes of IL-5 and 1 epitope of albumin derived from immunized phage display libraries. This trivalent IL-5-HSA Nb exhibited similar IL-5/IL-5R blocking activities to mepolizumab (Nucala), an approved targeting IL-5 mAb. Surprisingly, this trivalent Nb was 58 times more active than mepolizumab in inhibiting TF-1-cell proliferation. In primate studies, the trivalent IL-5-HSA Nb showed excellent pharmacokinetic properties, and peripheral blood eosinophil levels remained significantly suppressed for two months after a single dose. In addition, the trivalent IL-5-HSA Nb could be produced on a large scale in a P. pastoris X-33 yeast system with high purity and good thermal stability. CONCLUSIONS: These findings suggest that the trivalent bispecific IL-5-HSA Nb has the potential to be a next-generation therapeutic agent targeting IL-5 for the treatment of severe eosinophilic asthma.

Acceptor Planarization and Donor Rotation: A Facile Strategy for Realizing Synergistic Cancer Phototherapy via Type I PDT and PTT.

Tumor hypoxia seriously impairs the therapeutic outcomes of type II photodynamic therapy (PDT), which is highly dependent upon tissue oxygen concentration. Herein, a facile strategy of acceptor planarization and donor rotation is proposed to design type I photosensitizers (PSs) and photothermal reagents. Acceptor planarization can not only enforce intramolecular charge transfer to redshift NIR absorption but also transfer the type of PSs from type II to type I photochemical pathways. Donor rotation optimizes photothermal conversion efficiency (PCE). Accordingly, three 3,6-divinyl-substituted diketopyrrolopyrrole (DPP) derivatives, 2TPAVDPP, TPATPEVDPP, and 2TPEVDPP, with different number of rotors were prepared. Experimental results showed that three compounds were excellent type I PSs, and the corresponding 2TPEVDPP nanoparticles (NPs) with the most rotors possessed the highest PCE. The photophysical properties of 2TPEVDPP NPs are particularly suitable for in vivo NIR fluorescence imaging-guided synergistic PDT/PTT therapy. The proposed strategy is helpful for exploiting type I phototherapeutic reagents with high efficacy for synergistic PDT and PTT.

Structures of a deltacoronavirus spike protein bound to porcine and human receptors.

Porcine deltacoronavirus (PDCoV) can experimentally infect a variety of animals. Human infection by PDCoV has also been reported. Consistently, PDCoV can use aminopeptidase N (APN) from different host species as receptors to enter cells. To understand this broad receptor usage and interspecies transmission of PDCoV, we determined the crystal structures of the receptor binding domain (RBD) of PDCoV spike protein bound to human APN (hAPN) and porcine APN (pAPN), respectively. The structures of the two complexes exhibit high similarity. PDCoV RBD binds to common regions on hAPN and pAPN, which are different from the sites engaged by two alphacoronaviruses: HCoV-229E and porcine respiratory coronavirus (PRCoV). Based on structure guided mutagenesis, we identified conserved residues on hAPN and pAPN that are essential for PDCoV binding and infection. We report the detailed mechanism for how a deltacoronavirus recognizes homologous receptors and provide insights into the cross-species transmission of PDCoV.

Lin28B-high breast cancer cells promote immune suppression in the lung pre-metastatic niche via exosomes and support cancer progression.

The formation of pre-metastatic niche is a key step in the metastatic burden. The pluripotent factor Lin28B is frequently expressed in breast tumors and is particularly upregulated in the triple negative breast cancer subtype. Here, we demonstrate that Lin28B promotes lung metastasis of breast cancer by building an immune-suppressive pre-metastatic niche. Lin28B enables neutrophil recruitment and N2 conversion. The N2 neutrophils are then essential for immune suppression in pre-metastatic lung by PD-L2 up-regulation and a dysregulated cytokine milieu. We also identify that breast cancer-released exosomes with low let-7s are a prerequisite for Lin28B-induced immune suppression. Moreover, Lin28B-induced breast cancer stem cells are the main sources of low-let-7s exosomes. Clinical data further verify that high Lin28B and low let-7s in tumors are both indicators for poor prognosis and lung metastasis in breast cancer patients. Together, these data reveal a mechanism by which Lin28B directs the formation of an immune-suppressive pre-metastatic niche.

Characterization, immunostimulatory and antitumor activities of a ß-galactoglucofurannan from cultivated Sanghuangporus vaninii under forest.

A biomacromolecule, named as ß-galactoglucofurannan (SVPS2), was isolated from the cultivated parts of Sanghuangporus vaninii under the forest. Its primary and advanced structure was analyzed by a series of techniques including GC-MS, methylation, NMR, MALS as well as AFM. The results indicated that SVPS2 was a kind of 1, 5-linked ß-Glucofurannan consisting of ß-glucose, ß-galactose and α-fucose with 23.4 KDa. It exhibited a single-stranded chain with an average height of 0.72 nm in saline solution. The immunostimulation test indicated SVPS2 could facilitate the initiation of the immune reaction and promote the secretion of cytokines in vitro. Moreover, SVPS2 could mediate the apoptosis of HT-29 cells by blocking them in S phase. Western blot assay revealed an upregulation of Bax, Cytochrome c and cleaved caspase-3 by SVPS2, accompanied by a downregulation of Bcl-2. These results collectively demonstrate that antitumor mechanism of SVPS2 may be associated with enhancing immune response and inducing apoptosis of tumor cells in vitro. Therefore, SVPS2 might be utilized as a promising therapeutic agent against colon cancer and functional food with immunomodulatory activity.

Long noncoding RNA Meg3 sponges miR-708 to inhibit intestinal tumorigenesis via SOCS3-repressed cancer stem cells growth.

BACKGROUND: Colorectal cancer (CRC) remains the most common gastrointestinal cancer and a leading cause of cancer deaths worldwide, with most showing pathologies indicating the malignant transformation of early stage intestinal stem cells. The long non-coding RNA Meg3, which functions as a tumor suppressor, has been reported to be abnormal in multiple tumorigenesis events; however, the underlying mechanism by which Meg3 contributes to the malignant proliferation of colonic stem cells remains unclear. METHODS: We analyzed the expression levels of Meg3, miR-708, and SOCS3 in samples from Apc loss-of-function (Apcmin) mice and patients with CRC, particularly in colonic crypt cells. Apcmin mice and AMO/DSS-induced mice model (in vivo) and organoid culture system (in vitro) were used to explore the effect of the Meg3/miR-708/SOCS3 axis on tumorigenesis in the colon. In vitro, we performed RNApull-down, RNA immunoprecipitation, and luciferase reporter assays using DLD1 and RKO cell lines. FINDINGS: The Meg3/miR-708/SOCS3 signaling axis plays a critical role in the early stage of CRC development. Our data showed Meg3 levels negatively correlate with miR-708 levels both in clinical samples and in the Apcmin mouse model, which indicated that Meg3 acts as a competitive endogenous RNA (ceRNA) of miR-708. Then, miR-708 served as an oncogene, inducing neoplasia in both Apcmin mice and cultured colonic organoids. Put together, miR-708 appears to promote malignant proliferation of colonic stem cells by targeting SOCS3/STAT3 signaling. INTERPRETATION: These data revealed that Meg3 sponges miR-708 to inhibit CRC development via SOCS3-mediated repression of the malignant proliferation of colonic stem cells. The Meg3/miR-708/SOCS3 signaling axis provides potential targets for the diagnosis and treatment of CRC, particularly early stage CRC.

Extract From Tetrastigma hemsleyanum Leaf Alleviates Pseudomonas aeruginosa Lung Infection: Network Pharmacology Analysis and Experimental Evidence.

Tetrastigma hemsleyanum Diels & Gilg (T. hemsleyanum) has attracted much attention due to its ability on pneumonia, bronchitis, and immune-related diseases, while its functional components and underlying mechanism of action on pneumonia have not been fully elucidated. Herein, we used a systematic network pharmacology approach to explore the action mechanism of T. hemsleyanum leaf in the treatment of pneumonia. In this study, the results of network pharmacology demonstrated that there were 34 active components and 80 drug-disease targets in T. hemsleyanum leaf, which were strongly in connection with signal transduction, inflammatory response, and the oxidation-reduction process. Subsequently, a mouse model of pneumonia induced by Pseudomonas aeruginosa (P. aeruginosa) was established to validate the predicted results of network pharmacology. In the animal experiments, aqueous extract of T. hemsleyanum leaf (EFT) significantly attenuated the histopathological changes of lung tissue in P. aeruginosa-induced mice and reduced the number of bacterial colonies in BALFs by 96.84% (p < 0.01). Moreover, EFT treatment suppressed the increase of pro-inflammatory cytokines IL-17, IL-6, and TNF-α in lung tissues triggered by P. aeruginosa, which led to the increase of Th17 cells (p < 0.05). High concentration of EFT treatment (2.0 g/kg) obviously increased the anti-inflammatory cytokine levels, accompanied by the enhancement of Treg proportion in a dose-dependent manner and a notable reversal of transcription factor RORγt expression. These findings demonstrated that network pharmacology was a useful tool for TCM research, and the anti-inflammatory effect of EFT was achieved by maintaining Th17/Treg immune homeostasis and thereby suppressing the inflammatory immune response.

Probing into the In-Situ Exsolution Mechanism of Metal Nanoparticles from Doped Ceria Host.

Exsolved nanoparticle catalysts have recently attracted broad research interest as they simultaneously combine the features of catalytic activity and chemical stability in various applications of energy conversion and storage. As the internal mechanism of in-situ exsolution is of prime significance for the optimization of its strategy, comprehensive research focused on the behaviors of in-situ segregation for metal (Mn, Fe, Co, Ni, Cu, Ag, Pt and Au)-substituted CeO2 is reported using first-principles calculations. An interesting link between the behaviors of metal growth from the ceria host and their microelectronic reconfigurations was established to understand the inherent attribute of metal self-regeneration, where a stair-stepping charge difference served as the inner driving force existing along the exsolving pathway, and the weak metal-coordinate associations synergistically facilitate the ceria's in-situ growth. We hope that these new insights provide a microscopic insight into the physics of in-situ exsolution to gain a guideline for the design of nanoparticle socketed catalysts from bottom to top.

A potent neutralizing nanobody against SARS-CoV-2 with inhaled delivery potential.

The coronavirus disease 2019 (COVID-19) pandemic has become a serious burden on global public health. Although therapeutic drugs against COVID-19 have been used in many countries, their efficacy is still limited. We here reported nanobody (Nb) phage display libraries derived from four camels immunized with the SARS-CoV-2 spike receptor-binding domain (RBD), from which 381 Nbs were identified to recognize SARS-CoV-2-RBD. Furthermore, seven Nbs were shown to block interaction of human angiotensin-converting enzyme 2 (ACE2) with SARS-CoV-2-RBD variants and two Nbs blocked the interaction of human ACE2 with bat-SL-CoV-WIV1-RBD and SARS-CoV-1-RBD. Among these candidates, Nb11-59 exhibited the highest activity against authentic SARS-CoV-2 with 50% neutralizing dose (ND50) of 0.55 µg/ml. Nb11-59 can be produced on large scale in Pichia pastoris, with 20 g/L titer and 99.36% purity. It also showed good stability profile, and nebulization did not impact its stability. Overall, Nb11-59 might be a promising prophylactic and therapeutic molecule against COVID-19, especially through inhalation delivery.

Mechanism of upconversion luminescence enhancement in Yb3+/Er3+ co-doped Y2O3 through Li+ incorporation.

Li+ doping is a well-known, simple, yet efficient strategy to optimize the properties of upconverting materials. Nonetheless, the position of Li+ in the lattice and the mechanism of upconversion enhancement are still controversial, especially in Yb3+/Er3+ co-doped Y2O3. This paper presents a comprehensive investigation of the above issues (i.e. the position occupied by Li+ in the lattice and the mechanism of luminescence enhancement, in terms of decreased defects) by studying (Y0.78-XYb0.20Er0.02LiX)2O3 powders. Neutron powder diffraction was employed for the first time in the literature to show that Li+ ions are accommodated in Y sites of YO6 octahedra, confirmed also by the content of oxygen defects, which was increased with the increase of Li+ concentration. FT-IR showed that there was a small change in the amount and the type of the surface-absorbed groups with the increase in the Li+ content, thus not supporting the prevailing conclusion that the quenching groups are decreased by doping Li+. Positron annihilation lifetime (PLAS) experiments showed that the total defect concentration and the large defect clusters, which are considered as quenching centers, are decreased with increasing Li+-content, resulting in the enhancement of the emission intensity in Yb3+/Er3+ co-doped Y2O3.

Loss of Scribble confers cisplatin resistance during NSCLC chemotherapy via Nox2/ROS and Nrf2/PD-L1 signaling.

BACKGROUND: Cisplatin resistance remains a major clinical obstacle to the successful treatment of non-small cell lung cancer (NSCLC). Scribble contributes to ROS-induced inflammation and cisplatin-elevated toxic reactive oxygen species (ROS) promotes cell death. However, it is unknown whether and how Scribble is involved in the cisplatin-related cell death and the underlying mechanism of Scribble in response to chemotherapies and in the process of oxidative stress in NSCLC. METHODS: We used two independent cohorts of NSCLC samples derived from patients treated with platinum-containing chemotherapy and xenograft modeling in vivo. We analyzed the correlation between Scribble and Nox2 or Nrf2/PD-L1 both in vivo and in vitro, and explored the role of Scribble in cisplatin-induced ROS and apoptosis. FINDINGS: Clinical analysis revealed that Scribble expression positively correlated with clinical outcomes and chemotherapeutic sensitivity in NSCLC patients. Scribble protected Nox2 protein from proteasomal degradation. Scribble knockdown induced cisplatin resistance by blocking Nox2/ROS and apoptosis in LRR domain-dependent manner. In addition, low levels of Scribble correlated with high levels of PD-L1 via activation of Nrf2 transcription in vivo and in vitro. INTERPRETATIONS: Our study revealed that polarity protein Scribble increased cisplatin-induced ROS generation and is beneficial to chemotherapeutic outcomes in NSCLC. Although Scribble deficiency tends to lead to cisplatin resistance by Nox2/ROS and Nrf2/PD-L1, it is still possible that Scribble deficiency-induced PD-L1 may yield benefits in immunotherapy. FUND: National Key R&D Program of China, Strategic Priority Research Program of the Chinese Academy of Sciences, National Natural Science Foundation of China, China Postdoctoral Science Foundation.

Total flavonoids from Tetrastigma hemsleyanum ameliorates inflammatory stress in concanavalin A-induced autoimmune hepatitis mice by regulating Treg/Th17 immune homeostasis.

OBJECTIVE: Tetrastigma hemsleyanum, a rare and endangered medicinal plant, has attracted much attention due to its immunoregulatory and hepatoprotective activities. This study aimed to evaluate the anti-inflammatory effects and underlying mechanisms of total flavonoids from T. hemsleyanum(TFT)on Con A-induced hepatitis in mice. METHODS: TFT (1, 2 and 4 g/kg) and a positive control drug bifendate (200 mg/kg) were administered intragastrically to mice once daily for 10 consecutive days. On the 10th day, the model autoimmune of hepatitis was established by intravenous injection of Con A (20 mg/kg) 1 h after drug administration. Liver injury was assessed by serum levels of alanine amino transferase (ALT and AST) and histopathology 8 h after Con A injection. The levels of pro-inflammatory Th17 cytokines (IL-17, IL-6) and anti-inflammatory Treg cytokines (IL-10, TGF-ß1) in serum were evaluated by ELISA, the levels of Th17 and Treg cells infiltrated into spleen were investigated by flow cytometry methods, and hepatic tissue transcription factor Foxp3 and RORγt mRNA were determined using quantitative real-time PCR. RESULTS: Pretreatment with TFT and bifendate significantly reduced the serum levels of ALT and AST, and attenuated histopathological alterations in Con A-induced liver injury. With respect to samples treated with Con A alone, TFT and bifendate pretreatments differentially attenuated the increase of serum inflammatory factors interleukin (IL)-17 and IL-6 levels, the proportions of Th17 cells in spleen and the expression of RORγt in hepatic tissues. Meanwhile, TFT and bifendate pretreatments could enhance the percentage of Treg cells in spleen and the expression of Foxp3 in hepatic tissues, as well as the levels of transforming growth factor (TGF)-ß1, IL-10 in serum. CONCLUSION: The anti-inflammatory effects of TFT was mediated by regulating Treg/Th17 immune homeostasis, which, therefore, suppressed the inflammatory immune response. This study provided scientific basis for the further researches and clinical applications of Tetrastigma hemsleyanum.