The liver is perfused by both arterial and venous blood, with a resulting abnormal microenvironment selecting for more-aggressive malignancies. Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer, the sixth most common cancer globally, and the third leading cause of cancer-related mortality worldwide. HCC is characterized by its hypervascularization. Improving the efficiency of anti-angiogenic treatment and mitigation of anti-angiogenic drug resistance are the top priorities in the development of non-surgical HCC therapies. Endoglin (CD105), a transmembrane glycoprotein, is one of the transforming growth factor β (TGF-β) co-receptors. Involvement of that protein in angiogenesis of solid tumours is well documented. Endoglin is a marker of activated endothelial cells (ECs), and is preferentially expressed in the angiogenic endothelium of solid tumours, including HCC. HCC is associated with changes in CD105-positive ECs within and around the tumour. The large spectrum of endoglin effects in the liver is cell-type- and HCC- stage-specific. High expression of endoglin in non-tumour tissue suggests that this microenvironment might play an especially important role in the progression of HCC. Evaluation of tissue expression, as well as serum concentrations of this glycoprotein in HCC, tends to confirm its role as an important biomarker in HCC diagnosis and prognosis. The role of endoglin in liver fibrosis and HCC progression also makes it an attractive therapeutic target. Despite these facts, the exact molecular mechanisms of endoglin functioning in hepatocarcinogenesis are still poorly understood. This review summarizes the current data concerning the role and signalling pathways of endoglin in hepatocellular carcinoma development and progression, and provides an overview of the strategies available for a specific targeting of CD105 in anti-angiogenic therapy in HCC. Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer, the sixth most common cancer globally, and the third leading cause of cancer-related mortality in both sexes worldwide, with increasing incidence and mortality ,2. MolecHCC is one of the highly vascularized solid tumours, with angiogenesis playing an important role in its development, growth rate, and prognosis ,5. Many Due to the observation that endoglin CD105) is selectively expressed (or overexpressed) in activated vascular ECs in tumours (including HCC), it was hypothesized that it can also be a useful target for vascular-targeted anti-angiogenic therapy 5 is sele,12,13 anThe role of TGF-β systems (including endoglin) in carcinogenesis of solid tumours was well described in many works. However, none of them focused on HCC in particular . The molCurrently, most works describing the role of factors stimulating the angiogenic process in human cancers (including HCC) were focused on VEGF ,8,25. ThTo our knowledge, there is not yet a review that summarizes the results of basic and clinical studies that investigate the role of endoglin in HCC progression. Hence, an attempt to fill this gap was made in this manuscript, choosing endoglin as its focus for several reasons: (1) expression of that glycoprotein represents the proliferation status of liver sinusoidal ECs (LSECs), which are key in angiogenesis of the HCC microenvironment; (2) endoglin seems to be a better quantitative marker of microvessels density (MVD) compared to other microvessel markers e.g., CD34 , which aThus, in this review, we have summarized recent studies of endoglin functions and related signalling pathways as a diagnostic-prognostic biomarker of HCC and a potential molecular target of anti-angiogenic therapy. The available data on the role of endoglin in angiogenesis were updated, with an attempt to explain the mechanisms of CD105 action in the CD105 microenvironment of HCC in humans. In the review of works describing anti-angiogenic therapies, we have particularly focused on manuscripts that analyse endoglin as a target, and which could have applications in HCC therapy. Angiogenesis, a multi-stage process of blood vessel formation, is closely associated with neoplasia. The vascular network plays a major role in the creation of an immunosuppressive environment that allows tumours to “escape” from the immune system effects. New blood vessels are a proxy that facilitates cancer cell expansion, as well as tumour microenvironment maintenance ,26. On tThe role of endoglin in tumour-associated angiogenesis, as well as recent tests of anti-endoglin therapies, need to be briefly related to two other pro-angiogenic factors which are closely associated with ECs activation. Historically, the first recognised factor of this type was tumour-angiogenesis factor (TAF), discovered in the 1970s . The isoThe second factor was VEGF, the discovery of which also proved to be a scientific and therapeutic breakthrough, as it marked the beginning of a new era in anti-angiogenic therapy of solid tumours in humans (including HCC) ,5,6,7. CThe bases of current knowledge of solid tumour angiogenesis (including HCC) were undoubtedly laid by Judah Folkman, who first emphasized the importance of tumour vascularity for tumour growth at the beginning of the 1970s ,27. CallThus, another more specific pro-angiogenic factor was first discovered by the group of Senger et al. and initially called Vascular Permeability Factor (VPF) (currently VEGF). This factor was isolated from both the culture medium and ascites fluid of one tumour, the guinea pig line 10 hepatocarcinoma as a protein of 34–42 kDa in size, which differed from the permeability factors that were known at the time . CurrentAnother protein linked to the vascular endothelium was discovered in the 1990s. Initially called the 44G4 antigen, it was composed of two subunits of 95 kDa molecular weight, linked by disulphide bond(s) . Today, Similar to VEGF, endoglin expression is also detected on cellular membranes and in the cytoplasm of non-endothelial cells of normal and malignant tissues ,46,47,48In summary, all three of the described factors are glycoproteins that share, above all, their endothelial cell-specific mitogenic activities (pro-angiogenic factors). This activity mostly occurs in conditions of long-term tissue hypoxia, which accompanies neoplasia. Research articles suggest that these proteins tightly cooperate in the angiogenic process, taking complex responsibility for the quantitative and qualitative changes of the tumour vasculature. The change of blood vessel phenotype involves the appearance of new endothelial tumour markers, e.g., VEGFRs, and/or endoglin. While the signalling pathways of VEGF and endoglin differ, the final effect of the action of both of these factors is pro-angiogenic signalling. Detailed mechanisms of endoglin as a membranous EC marker were described in the further parts of this manuscript. All three of the mentioned proteins can be produced by different cell types, including: (1) tumour cells , (2) endothelial cells , (3) numerous non-endothelial cells . The subcellular location of these proteins also differs; VEGF is largely present in the cytoplasm, while endoglin is mostly a membranous protein. It is worth noting that increased endoglin expression can be observed in proliferating tumour ECs, with only small expression in resting ECs, which might be an advantage in the choice of anti-endoglin therapy. HCC is one of the most vascular solid tumours, in which angiogenesis plays an important role ,5,54,55.HCC is characterized by its hypervasculature resulting from higher expression of angiogenesis promoting factors, especially VEGF and VEGF-R, but also other pro-angiogenic factors, such as angiopoietin 2, PDGF, EGF, FGF, TGF-α, and endoglin ,59,60,61The liver is perfused by both arterial and venous blood, with the resulting abnormal microenvironment selecting for more-aggressive malignancies . Liver sThe participation of the BM-EPC subpopulation in HCC angiogenesis is considered uncertain by one of the authors ,64 but oEndoglin (CD105) is a type I integral membrane-bound glycoprotein that serves as a co-receptor (with betaglycan) for members of the transforming growth factor-β (TGF-β) superfamily of proteins which has a crucial role in fibrogenesis and angiogenesis ,68,69,70Endoglin is a 180 kDa homodimer and consists of a large extracellular domain (561 aa), regular hydrophobic transmembrane domain, and a short serine/threonine-rich cytoplasmic tail ,68. It oIn humans, endoglin is expressed in the liver with a much higher abundance of the L-ENG splice variant . The resEndoglin also occurs in a soluble form (Sol-ENG), which was detected in the serum of healthy as well as diseased patients (including those diagnosed with HCC) ,40,72,82Endoglin engages in many protein complexes, including those with TGF-β superfamily members: TGF-β1 and TGF-β3 isoforms, type-I and type-II TGF-β serine/threonine kinase receptors , and other cytoplasmic proteins . EndogliGenetic and biochemical studies of quiescent ECs prove that endoglin is necessary for TGF-β/ALK1  signalling . In a noEndoglin, similar to other TGF-β receptors, is involved in modulating a response to the binding of TGF-β1 and TGF-β3 isoforms, activin-A, bone morphogenic proteins (BMPs) -2, -7, -9, -10, β-arrestin2, and α5β1 integrin ,91,92,93Endoglin may also regulate EC behaviour independently of TGF-β signalling by regulating cytoskeleton organization, protecting from apoptosis, stimulating c-Jun N-terminal kinase 1 (JNK1) phosphorylation, and regulating endothelial nitric oxide synthase (eNOS) expression ,89. EndoEndoglin gene mutation is responsible for the autosomal dominant type-1 hereditary haemorrhagic telangiectasia . The most prominent symptoms of HHT1 are capillary dysplasia and recurrent haemorrhage , with chSoluble endoglin is also involved in the etiopathogenesis of preeclampsia. In this disease, increased expression of Sol-ENG by the placenta, and a decreased expression of VEGF-A and PlGF cause impaired angiogenesis and vasculogenesis. An intense inflammatory response, EC injury, generalized vascular resistance, and disseminated intravascular coagulation were described as symptoms ,99. Endoglin is an important marker of angiogenesis in the case of many carcinomas, including HCC ,100,101.Depending on the type of a tumour and endoglin producing cells (vascular ECs vs. tumour cells), this protein can serve dual roles (growth/tumour progression or inhibition/tumour suppression) in carcinogenesis . HenThe release of circulating endoglin, Sol-ENG, produced by ECs as well as neoplastic cells, was previously described ,23,40. TThe most important properties of endoglin, with particular attention to its role in the physiology and pathology of the liver, are presented in TGF-β1 via SMADs, hypoxia via HIF-1, and vascular injury via Kruppel-Like Factor 6 (KLF6) all upregulate endoglin transcription . Both hyOther regulators of endoglin expression in various cell types are well summarized in the literature (reviewed in ). Numerous studies indicate the participation of canonical and non-canonical TGF-β pathways in the process of fibrosis of various organs (including liver) ,89,119. Experiments on different models (in vitro and in two different in vivo models of liver fibrosis) prove that the endoglin expression increases through the process of HSCs transdifferentiation into MFBs ,121. MeuClinical studies bring up the fact of higher serum Sol-ENG levels in HCC patients with coexisting liver cirrhosis, as well as positive correlations of this protein’s serum levels with serum AFP levels. Some of them even consider Sol-ENG level as an independent marker  for the development of HCC in cirrhotic patients . Higher Summarizing the results of experimental and clinical studies, it seems that endoglin serves mostly a cytoprotective function in the progress of liver fibrosis. At the same time, evaluation of its tissue expression, as well as Sol-ENG serum concentration, might serve to grade the severity of histological changes in livers of patients with an isolated form of fibrosis, as well as fibrosis associated with chronic liver diseases (including HCC). More particular pathomechanisms of liver fibrosis involving endoglin are described in a later part of this work. The main molecules promoting angiogenesis in HCC are well known and described. They involve families of factors such as VEGF, angiopoietins, epidermal growth factors (EGFs), Platelet-Derived Endothelial Growth Factors (PD-ECGFs), or basic Fibroblast Growth Factors (bFGFs) ,124. TheThe studies investigating the role of endoglin in carcinogenesis of tumours (including HCC) can be divided into two groups: (1) those focused on roles of endoglin as a marker of angiogenesis and its role in diagnosis/prognostics ,40,100, In the first group of studies, the newest  available meta-analysis of data from 34 works indicates that high MVD-CD105 is a predictor of the poor OS, DFS, and CSS  . HoweverTissue expression of CD105 in vascular ECs  was detected by most of the authors ,20,21,22In HCC tissues, three patterns of CD105 expression on ECs, namely, sinusoid-like, branching, and small endothelial sprouts, first described by Ho et al., are most commonly cited . This stThe most recent studies generally confirm the observations concerning higher CD105 expression in NT tissue, as compared with HCC. Considering the TMA (90 patients), elevated expression of endoglin in all of a tumour adjacent tissues was observed, while in HCC only ~56% of the affected differentially expressed CD105 . The intensity of CD105 expression was significantly higher in NT tissue compared to HCC. CD105 glycoprotein was mainly located in the ECs. However, similarly to the findings of other authors , it was Most of the authors agree that the endoglin is an ideal instrument to study HCC microvessel density, i.e., it is better than the use of pan-endothelial EC markers , as it visualises the ECs of newly formed tumour vasculature and correlates with VEGF expression ,15,16. ASimilarly to other solid tumours of the gastrointestinal tract , HCC alsThere are also some results that note negative correlations between MVD-CD105 expression in tumour tissue and prognosis in HCC. Comparative studies of CD105 expression between different regions of HCC and control liver have shown that lowered expression of CD105 in ECs of tumour tissue, and more abundant expression in LSECs in NT tissues (including cirrhosis), might have prognostic significance in this type of cancer ,22,104. The most recent research based on tissues of HCC patients treated with sunitinib  showed that tissue expression of CD105 in tumour tissue monocytes, as well as a serum concentration of CD105, might serve as an independent predictive factor of the OS and progression-free survival (PFS) of advanced HCC. Interestingly, patients with lower endoglin expression or serum levels responded better to therapy with that drug .There are not many studies on the role of endoglin as a serum biomarker in HCC. The present results confirm the elevated levels of Sol-ENG in cirrhotic patients (5.8 µg/L) as compared with healthy (3.7 µg/L) or non-liver-disease controls (3.9 µg/L). The highest concentration of serum CD105 (7.4 µg/L) was observed in patients affected with HCC and cirrhosis, compared to liver cirrhosis only (5.8 µg/L) and HCC only (5.0 µg/L). In a group exhibiting the highest serum CD105 levels, serum AFP levels showed a high correlation. These authors consider Sol-ENG as an independent marker (OR 1.3) for the development of HCC in cirrhotic patients . SimilarIncreased endoglin and TGF-β mRNA expression were also demonstrated in the serum of HCC patients with coexisting cirrhosis, as well as cirrhotic patients, compared to control , but without a major difference between the diseased groups. Endoglin mRNA concentrations correlated positively with TNM clinical stage . In turnSummarizing the cited research, endoglin overexpression is mostly observed in proliferating ECs of small vascular and lymphatic vessels in cancer tissues, on different stages of solid tumour carcinogenesis. The studies on the potential role of endoglin as a tissue prognostic marker of HCC progression yield varying results.Some of the publications describe a positive correlation between CD105 expression in tumour tissue and clinical-pathological data and/or prognosis in HCC, similarly to other tumours of the gastrointestinal tract. However, CD105 positive immunostaining in non-tumour tissues (including cirrhosis) and/or control liver was not always detected, or not studied. Another group of reports considers the prognostic significance of increased tissue CD105 expression in non-tumour liver tissue (including cirrhosis), and lowered expression in the tissues of a tumour itself. The lower expression of CD105 in HCC, compared to non-tumour tissue and/or control, is usually described in larger, more aggressive tumours with portal veins invasion, poor differentiated HCC, and tumours with the more advanced TNM stage. According to some of the authors, the presence of higher CD105 expression in non-tumour tissue suggests that CD105 is not a good target of anti-angiogenic therapy in HCC, especially in patients with coexisting liver cirrhosis, as it might promote HCC development. There are also studies reporting the comparable expression of CD105 in HCC and non-tumour tissue. Lack of significant differences might be caused by an insufficient number of studied samples or by coexisting viral infections, such as HBV, HCV, that modulate the expression of pro-angiogenic factors, including endoglin. The remaining differences in the results of the analysed works are mostly caused by the diversity of technical approaches, variation in tissue pre-treatment protocols, different types of anti-endoglin mAb used in immunohistochemistry, and non-standardized counting methods.Singular studies are focused on the analysis of serum levels of Sol-ENG and/or CD105 mRNA level in HCC. These works mostly describe the increased concentrations of this form of endoglin in liver cirrhosis and/or HCC, correlating with some clinicopathological data. They suggest the consideration of Sol-ENG as a useful biomarker in HCC diagnostics. However, the sensitivity of Sol-ENG as an independent diagnostic-prognostic marker seems to be relatively weak. In prognostics, as a well as anti-angiogenic treatment initiation or effect monitoring, it seems important to connect the cellular level research  with estimation of levels of serum Sol-ENG (or endoglin mRNA) and/or other markers of HCC advancement.The results of the studies of the cited authors are compiled in Yu et al., verified the hypothesis that non-tumour (NT) tissue surrounding the HCC might be a hypoxic and highly angiogenic area, into which many more EPCs are recruited and homed ,31. It wIn turn, it was observed in tissue HCC material that the NT region shows high expression of different angiogenic factors (activator and inhibitor molecules) (reviewed in ), which In conclusion, the above-mentioned research indicates a potential role of EPCs and their differentiation into tumour ECs (expressing CD105 on their surface) in the process of HCC neovascularization. Increased expression of angiogenic factors (including endoglin) in the NT environment suggests that this region of the liver has a key role, especially in initial phases of liver carcinogenesis. Participation of endoglin CD105), as a proliferation-associated endothelial cell adhesion molecule included in the neovascularization process of HCC, is indisputable ,5,31,62., as a prIn recently conducted studies on morphology, phenotype, genotype and function of TECs derived from clinical HCC, the expression of numerous vascular markers (including CD105), as well as genes responsible for angiogenesis, were detected. These cells presented endothelial phenotype and morphologic features of ECs, and were capable of tube formation and migration . DiffereThe CD105 expression is also demonstrated in normal hepatocytes in the NT area , as wellA role of other CD105-positive liver cells in HCC pathogenesis cannot be ruled out. These include the previously mentioned HSCs , human lTGF-β, together with its endoglin co-receptor, is engaged in the processes of angiogenesis, which was presented on in vitro models and works with experimental animal models ,130,132.Using the model of cancer stem-like cell (CSCs) generated from HCV-infected primary and transformed human hepatocytes, it was shown that endoglin is upregulated (250-fold) in sphere-forming cells compared to primary hepatocytes . The samThere is also some proof of a link between hypothyroidism and elevated risk for HCC. In the endoglin gene promoter, the presence of thyroid hormone response elements (TREs) was also reported . The stuModulatory influence of endoglin on the inflammatory process was also described. In the murine model, the regulatory role of endoglin in leukocyte transendothelial migration, facilitated by binding to the α5β1 integrin (via its RGD motif) after stimulation by the CXCL12 inflammatory chemokine, was also proven. Leukocyte adhesion was inhibited by Sol-ENG and synthetic RGD peptides . This reIn summary , the resConsidering the fact that HCC is one of the most vascularised solid tumours, evaluation of angiogenesis with the use of anti-endoglin mAb should be included in the routine diagnostics and prognostic procedures in this type of cancer. Evaluation of the MVD-CD105 in tissue material should become an inherent step in the staging of HCC. Finally, liver fibrosis (exhibiting CD105 overexpression in HSCs) and HCC (with CD105 overexpression in TECs) can be added to the list of potential therapeutic target diseases for CD105-specific therapy.Effective cancer therapies should be directed towards not only lowering/inhibition of pro-angiogenic factors, but also target factors of vascular functions . Anti-enIn the case of HCC, the stage of tumour advancement, type of vascularization, and molecular type of angiogenesis are all important parameters. A further challenge will be to find the right combination of anti-angiogenic/anti-vascular drugs and standard forms of radio- or chemotherapy . PreviouOne of the new ideas of HCC therapy is the use of a chimeric IgG1 anti-CD105 monoclonal antibody (TRC105) that binds human endoglin with high avidity. It inhibits angiogenesis and tumour growth by ECs growth inhibition, antibody-dependent cellular cytotoxicity (ADCC) and apoptosis, and complements VEGF inhibitors . Data ab131I-anti-endoglin mAb. The effects of this therapy included significant tumour-growth suppression and the remarkably decreased tumour weight in animals [131I-anti-endoglin mAb and 5-fluorouracil on the same HCC model appear to be promising. The tumour volume and weight decreased, and inhibition rate of the tumour growth was up to 77.1 ± 4.06% [One of the increasingly used methods of solid tumour therapy the is the administration of cytotoxic radioisotopes conjugated to antibodies. Studies on the treatment with endoglin-targeted radioimmunotherapy have been carried out on a murine model of HCC. HCC tumour SMMC7721-GFP xenograft-bearing mice were treated with  animals . New tre animals . Also, r ± 4.06% . The first human study TRC105 treatment was published in 2012. Patients with advanced cancers showed the safety profile and clinical effectiveness of TRC105 therapy ,145. AlsNew combinations of therapies, using TRC105 and axitinib or TRC105 with bevacizumab, are undergoing clinical trials in other cancers  ,149. FurIn summary, phase-I and -II clinical trials showed the insufficient efficacy of TRC105 monotherapy in HCC. While still unsatisfactory, better results were obtained in combination therapy of TRC105 with sorafenib. On the basis of preclinical tests and data from the other types of tumours, it may be expected that TRC105 therapy in combination with other promising agents, evaluated in phase-III trials as first-line and second-line treatment for HCC, will yield better results. Knowledge of liver sinusoidal ECs markers (including CD105) helped to identify novel targets of anti-angiogenic therapies and/or to understand adverse effects of some drugs used for such therapy ,44. Due The therapy of various cancers with the use of nanoparticles (NPs) has become a current focus of interest. The combination of these unique NPs, possessing different physical and chemical properties, with other substances or drugs, increases the efficacy and safety of anticancer therapies compared to conventional methods. NPs can be designed to achieve the physicochemical and biological properties that are necessary to reach tumour cells while saving the healthy surrounding tissue. The data describing NPs anticancer activity in HCC in vitro and in vivo are not abundant in quantity, in contrast to those of other cancers ,152. Eco-friendly gold NPs , synthesized by Enterococcus spp. bacteria showed cytotoxic activity against HepG2 cell line in vitro. The anticancer activity depends on the concentration of NPs, the higher one gives the higher activity. In this study, the high efficacy was compared to standard cyclophosphamide . Silver Non-metallic silica NPs were used to improve the effective therapy of liver cancer. The functionalized silica nanoparticles (SLNs) were conjugated with doxorubicin via disulphide bonds, using a CPLGLAGG peptide substrate which may be split by the MMP-2 protease. In vitro and in vivo studies in HepG2 cells and HepG2 tumour-bearing mice confirmed that overexpressed MMP2 protease in tumour tissue, together with intracellular glutathione, causes rapid release of doxorubicin, leading to inhibition of tumour growth . The lowIn HCC cases, the association of NPs with anti-CD105 antibodies is only applicable to tumour diagnostics. The first data about anti-CD105 antibodies conjugated with gold nanoparticles was presented in 2014 in the trial of biodistribution of this complex in mice tumours. There was no observed effect on CD105-dependent tumour uptake and the efficacy of tumour targeting . The comIn summary, reports describing the use of NPs in HCC therapy are not abundant. However, they indicate that the combination of anticancer drugs with NPs may be a sensible method for drug delivery, as well as prevention of drug resistance. However, these require confirmation in clinical trials in human HCC cases. Such experimental therapies are now only in the initial design phase and require a long time to reach the potential widespread use. No data was found in the literature on HCC therapy that combines anti-CD105 agents with NPs and other drugs.Regulation of the angiogenic process in HCC, as well as other solid tumours, mainly involves cells that produce pro- and anti- angiogenic factors. Endoglin, as an important TGF-β co-receptor, serves its main role as a pro-angiogenic factor, responsible for the proliferation and survival of ECs (anti-apoptotic action). 6 copies per cell) than the VEGFRs (<0.2 × 106 copies per cell) [Endoglin expression also positively correlates with VEGF. However, endoglin is expressed on a much higher level (up to 3 × 10er cell) . EndogliThe so far obtained results on the expression of endoglin in HCC are relatively sparse, and are often contradictory. Statistically-significant correlations were described between both increased and decreased levels of MVD-CD105 in the tumour tissue and degree of HCC differentiation, more advanced clinical stage of HCC, and/or poor prognosis. Comparing the quantitative expression of endoglins in tumour tissue and non-tumour tissue, the higher expression in microvessels of non-tumour tissue (including liver cirrhosis) (NT) was often noted. Some researchers consider the latter location as a high angiogenesis area, a niche of EC progenitors, or the location of HSC activation towards myofibroblasts; in short, a unique microenvironment for tumour metastasis and growth. Others attribute the high expression in NT tissue to the protective functions of the endoglin, pointing out that anti-CD105 therapy could further damage the liver in early stages of the disease. In vivo and in vitro studies of endoglin action mechanisms in HCC point out that in the liver, the effects of this protein are cell-specific, depending on the aetiology of HCC , HCC differentiation, and stage of advancement of the pathological changes . It could be confirmed that endoglin has pro-angiogenic effects in tumour ECs, while at the same time promoting or inhibiting the processes of liver fibrosis and carcinogenesis when produced by other cells. Endoglin was chosen as a target for anti-angiogenic therapy, based on the observations of overexpression of this glycoprotein mainly in the newly sprouted tumour blood vessels, and not in the healthy vessels in the rest of the organ. The superiority of anti-angiogenic strategies over standard chemotherapeutics is often stated. In the case of TRC105 use, a few side-effects of such therapy were reported. However, this topic was never brought up in the studies of HCC . DespiteIn future research joining the fields of basic sciences and biotechnology, it could be worth analysing the EC progenitors in the liver or determining more precisely of the roles of other cells that synthesise endoglin in hepatocarcinogenesis (including tumour cells). Additionally, to determine the quantitative expression of endoglin in different regions of the affected liver more precisely, verification of quantitative methods of MVD-CD105 evaluation, as well as the applied study protocols, is still needed. From a clinical point of view, the qualification criteria for patients set to undergo anti-angiogenic HCC therapy should be more particular and monitored using better methods, especially in those affected by co-existing chronic conditions  in which endoglin and TGF-β signalling pathways play a major role. Endoglin (CD105) is involved in the process of HCC neoangiogenesis  under intratumoral hypoxia and acidosis, the main important characteristics of the tumour microenvironment.Endoglin is expressed preferentially on activated liver sinusoidal ECs (LSECs) which are characterized by enhanced angiogenic activity, spontaneous motility, greater capacity for migration, and increased resistance to apoptosis in response to TGF-β1.In HCC, three patterns of the CD105 expression in ECs are most commonly described .Both CD105-positive LSAECs and CD105-positive non-ECs  play a role in the pathogenesis of HCC.The large spectrum of endoglin effects in the liver is cell-type and HCC stage-specific.High expression of endoglin in non-tumour tissue suggests that the microenvironment might play an especially important role in the progression of HCC.For a reliable assessment of the diagnostic and prognostic role of endoglin in HCC, simultaneous examination of different tumour areas, with a comparison to healthy control, unification of research protocols, and verification of MVD-CD105 counting systems, are required.Endoglin  tissue expression level examination should be combined with measurement of serum levels of Sol-ENG and other HCC biomarkers and/or determination of the histological stage of the tumour, to successfully predict HCC and effectively apply anti-CD105 therapy.Due to the high heterogeneity of pathological changes in HCC and tumour evolution, the effective anti-CD105 therapy in this cancer is not yet available, remaining a goal of the precision medicine development.The main mechanism that stimulates endoglin production in the HCC is the hypoxia of the tumour environment. However, influence of other factors was also proven .In HCC-associated angiogenesis in vivo, endoglin expression mainly concerns tumour ECs (TECs), activated ECs in adjacent non-tumour area, and different non-endothelial cells .In HCC, there is a tight relation between overproduction of endoglin by proliferation tumour ECs (TECs) and creation of sinusoid-like, branching, and small endothelial sprouts. The same relation is not observed between endoglin expression and vasculogenic mimicry.Higher endoglin expression in non-tumour area, as compared with tumour tissue, suggests that this location may play a very important role in the progression of HCC.The large spectrum of endoglin effects in the liver is cell-type specific, depends on aetiology (higher expression in HCV infection), and is HCC stage-specific (higher expression in HCC with cirrhosis).Due to the heterogeneity of HCC and multifactorial aetiology of the cancer, the satisfactory effects of anti-CD105 monotherapy (TRC105) are not yet available, remaining a goal of the precision medicine development.


This test could be easily used on site to detect heroin in oral fluid, and it could be a promising product in the future including for driving under the influence.We used lateral flow strips based on gold nanoparticles to detect 6-monoacetylmorphine  in oral fluid samples. In this competitive lateral chromatographic immunoassay, the 6-MAM was chemically synthesized and conjugated to bovine serum albumin. The results were qualitatively detected via the colour change of the test line. By using a proper sample pad, a suitable nitrocellulose membrane and a customized sponge device adsorbed the oral fluid directly from the mouth; the total test time was 3 min. The sensitivity of the assay was 4.0 ng ml According to the World Drug Report 2017 published by the United Nations Office on Drugs and Crime, the use of opiates and prescription opioids may not be as widespread as cannabis, but opioids remain major drugs with potential harm and health consequences [Illicit heroin abuse is one of the most common forms of opioid addiction. Heroin  is a semi-synthetic morphine derivative and a powerful opioid analgesic . The metSeveral methods to detect 6-MAM have been described, and these can be divided into the following categories: (1) chromatographic analysis including gas chromatography –7 and hiOf the methods for rapid detection, colloidal gold nanoparticle (AuNP)-based lateral flow strips (LFSs) have been widely adopted for rapid screening due to the size-dependent and distance-dependent optical property of AuNPs, with the first report by Mirkin and co-workers . The pri−1 [Samples include blood, plasma, urine, hair, oral fluids as well as in breath, sweat, breast milk, teeth, etc. –27. The −1 ,32. HereWe explored AuNPs as antibody labels in a lateral flow assay for rapid and sensitive detection of 6-MAM via a colorimetric signal. First, we synthesized the 6-MAM and then conjugated it to bovine serum albumin (BSA) to enable it to be coated on a T line. Moreover, to overcome the difficulties in dealing with oral fluid samples, the types of nitrocellulose (NC) membranes, the solution formula of the sample pad and the sponge adsorbent pad were chosen to search the best conditions for oral fluid LFSs. Finally, the 6-MAM LFS was validated and shown to have outstanding sensitivity and specificity.2.2.1.−1) was made by a RephiLe PURIST UV Ultrapure water system (China). The Reel dispersion system was from Doyesgo (China). The Vion IMS Q-Tof mass spectrometer was from Waters (USA). All standard materials such as 6-MAM and morphine were obtained from the National Institutes for Food and Drug Control (China). The microscope was from Motic AE2000 . All other chemical and immunological reagents not specified here were standard commercial products of analytical/reagent grade.The antibodies against 6-MAM were supplied by Bioventure (Shanghai). BSA and polyvinyl pyrrolidone (PVP) were purchased from Sigma . Triton X-100, Tetronic 1307 (S9), Ohodasurf On-870 (S17) and STANDAPOL ES-1 (S7) were purchased from BASF (Germany). Distilled water , a sample pad, a conjugated pad, NC membranes and an absorbent pad. The sponge adsorbent strip is specially designed for oral fluid collection and quickly transports oral fluid into the sample pad. The sample pad contains a buffer system and some surfactants. The antibody–AuNP conjugates were sprayed on the conjugate pad to react with sample and be released from the pad and enter the NC membrane coated with 6-MAM–BSA on the T line and goat anti-rabbit antibodies on the C line. The absorbent pad is a filter paper located at the end of the strip; it maintains the capillary flow. The LFS only needs to be placed into the mouth or inserted into an oral fluid sample cup. A schematic view of the LFS is shown in 2.3.N-hydroxysuccinimide (NHS) ester group was then added to the 6-MAM molecule to conjugate it to the carrier proteins . Finally, 500 µl of mixed AuNPs–antibody conjugates were sprayed on a 20 mm2 fibreglass and then dried at 37°C overnight.The same method was used to conjugate AuNPs to rabbit IgG antibodies. When making the conjugate pad, the antibody conjugates were diluted to absorbance five with buffer (0.05 M Tris-HCl containing 10.0 g l2.5.−1) were dispensed on the NC membranes as the test lines (T line). Control lines (C line) were coated by goat anti-rabbit polyclonal antibodies (0.15 mg ml−1). The coated NC membranes were dried at 37°C overnight. Nine commercial NC membranes from four companies were evaluated: Millipore , GE-Whatman (FF120HP and AE100), Sartorius (CN95 and CN150) and Pall (Vivid90 and Vivid170).To make the lateral flow test strip, 6-MAM–BSA antigens  for sensitivity detection. Ten commonly abused drugs were used to verify the specificity of the LFSs. These drugs were morphine , codeine , tetrahydrocannabinol , methylene dioxymethamphetamine , ketamine , methylamphetamine , cocaine , methadone , ephedrine  and pseudoephedrine .Negative oral fluid was collected from six people and spiked with 6-MAM  chromatograms of activated 6-MAM at 8.8 min with an 06.27645  versus aWe did not use gradient dimethylsulfoxide dialysis because the product is soluble and the previous conjugation protocol is too complex. BSA had several peaks in the UPLC chromatogram (data not shown) because of the various analogues. This led to a range of conjugation ratios. Therefore, there were various peaks in the UPLC chromatogram corresponding to the different conjugating ratios. The conjugation results could be more effectively confirmed via antigen–antibody pairing than the conjugating ratio.3.2.NC membranes bind proteins electrostatically via interactions of the strong dipole of the nitrate ester with the strong dipole of the peptide bonds of the protein. The properties including capillary flow rate, signal intensity and background were evaluated because they could influence the final performance of the LFS. Furthermore, the flow rate is given more attention because it could affect the protein adsorption capacity and even the sensitivity. The flow rate of a membrane depends on the aggregate properties of the porous structures such as pore size, pore size distribution and porosity. A larger pore size leads to weaker protein adsorption.−1. The signal intensity at the T line was also needed at the normal level. A deeper background colour hampered accuracy. The Millipore membrane HF135 was the best choice for 6-MAM after comprehensive consideration of sample flow rate, signal intensity at the T line and background colour.We compared nine NC membranes . Each te3.3.The solution for treating the sample pad is very important for the test because it served as the reaction buffer system when the oral fluid samples rehydrate the pad. The solution usually includes a buffer system with proper ionic strength and pH value; some blocking materials and surfactants can accelerate the oral fluid flow rate on the membrane. The sample pad deals with the complexities of the oral fluid's matrix effects and makes it compatible with the NC membrane. Moreover, the buffer system ensures the release of analytes and stabilizes the flow rate because the oral fluid is too viscous.−1, with a normal signal intensity and a white background. Surfactant S7 is a strong anionic surfactant that offers stronger washing capability than S17 and S9.Four different formulas were considered. The solution formulas are given in 3.4.Oral fluids are more difficult to collect than urine due to their high viscosity. There are many kinds of oral fluid-collecting devices: cotton swabs, sponges, plastic tubes and cups. Some methods stimulate oral fluids via vinegar, mouthwashes, lozenges, etc. However, such stimulation may change the concentration of analytes in the oral fluid and is more complicated and time-consuming. We finally collected oral fluid directly from the mouth by a sponge adsorbent , which is a blend of polymeric fibres with a suitable pore size.−1) than the K1 sponge for water, PBS buffer or real oral fluids. This is a very important issue for oral fluid testing. In conclusion, K2 was chosen for its distinguished performance in dealing with oral fluid samples.Two kinds of sponge adsorbents (K1 and K2) were custom-designed, and the structures of both sponge pads are shown in 3.5.−1, which meets the requirements for general oral fluid detection limits [Small molecules are usually detected via a competitive assay in LFSs. Here, there is no signal (red line) in the T line. This represented a concentration of 6-MAM in the sample that is above the cut-off value. The sensitivity for oral fluid testing should be much higher than the urine test because of the low concentration of drug metabolites in oral fluid. Here, we successfully made a qualitative LFS for 6-MAM with a sensitivity of 4 ng mln limits ,29. The 4.The 6-MAM is the specific metabolite of heroin. We report here an LFA for 6-MAM via a special conjugate paired with a specific antibody. We made a conjugate that linked 6-MAM to the carrier protein via an NHS ester group at the C3 position . Here, tWith the above advantages, the 6-MAM LFS for oral fluid sample could be applied to both research and industry uses. It could help the police save manpower and time/costs in preliminary screening. Oral fluids are convenient and less invasive and are suitable for traffic screening. In conclusion, an oral fluid LFS for heroin abuse aimed at 6-MAM is a promising product to combat drugged driving.


Trp53 (but not Tgfbr2) deletion in endothelial cells (ECs) inhibited radiation-induced EndMT, reducing tumour regrowth and metastases with a high CD44v6+ cancer-stem-cell (CSC) content after radiotherapy. Osteopontin, an EndMT-related angiocrine factor suppressed by EC-Trp53 deletion, stimulated proliferation in dormant CD44v6+ cells in severely hypoxic regions after radiation. Radiation-induced EndMT significantly regulated tumour-associated macrophage (TAM) polarization. CXCR4 upregulation in radioresistant tumour ECs was highly associated with SDF-1+ TAM recruitment and M2 polarization of TAMs, which was suppressed by Trp53 deletion. These EndMT-related phenomena were also observed in irradiated human lung cancer tissues. Our findings suggest that targeting tumour EndMT might enhance radiotherapy efficacy by inhibiting the re-activation of dormant hypoxic CSCs and promoting anti-tumour immune responses.It remains controversial whether targeting tumour vasculature can improve radiotherapeutic efficacy. We report that radiation-induced endothelial-to-mesenchymal transition (EndMT) leads to tumour vasculature with abnormal SMA Radiotherapy is the main treatment for most cancer, but it is unclear if targeting tumour vasculature can enhance tumour radiosensitivity. Here, the authors show that tumour endothelial-mesenchymal transition after radiotherapy leads to proliferation of radioresistant CSCs and tumour associated macrophages polarization. Multiple strategies for targeting cancer cells, cancer stem cells (CSCs), tumour stroma, and tumour endothelial cells (ECs), as well as improving anti-tumour immune responses to increase tumour radiosensitivity, are being developed4. Several anti-angiogenics have been clinically evaluated in combination with radiotherapy6; however, their benefits are controversial. Bone marrow-derived cell (BMDC) recruitment to irradiated tumours may contribute to tumour relapse via vasculogenesis8. Although tumour-vasculature development after radiotherapy is not well characterized, targeting tumour ECs enhances radiotherapeutic efficacy; ceramide, sphingomyelinase, and Bax regulate EC apoptosis after irradiation10. Vascular damage may affect tumour responses to high radiation doses, e.g., during stereotactic radiosurgery/radiotherapy12. ECs lacking ataxia-telangiectasia mutated showed increased radiosensitivity13. However, it remains debatable whether EC targeting can improve radiotherapy efficacy.Anti-angiogenic or vascular-destructive agents potentially enhance tumour responses to radiotherapy14. CSCs are often quiescent after radiation or chemotherapy and their awakening causes tumour relapse and escape16. Understanding the mechanism regulating the dormant or proliferative status better is important for targeting CSCs.Cancer cells that acquire radioresistance exhibit CSC-like characteristics17. Radiotherapy can enhance immunosuppressive responses, including chemotactic signals that recruit several myeloid cell types17. Radio-immunomodulation studies have revealed crucial strategies for effectively combining immunotherapy and radiotherapy.Radiotherapy can stimulate anti-tumour immune responses. Immunomodulation using antibodies against programmed death 1 and programmed death-ligand 1 in combination with radiotherapy has been assessed in clinical trials18, affects the endothelium to enable tumour-cell extravasation19, and may give rise to pericyte-like cells within tumours20. Pericytes play critical roles in blood-vessel maturation and blood-barrier maintenance and regulate vessel integrity and function by interacting with ECs22. Tumour vessels harbouring less pericytes are more sensitive to radiation and chemotherapy23.Endothelial-to-mesenchymal transition (EndMT) promotes cancer-associated fibroblast formation in tumoursHere, we studied tumour EndMT and pericyte-derived tumour vasculature during tumour regrowth after radiotherapy. We analysed the effects of EndMT-regulated vasculature on the irradiated tumour microenvironment, especially, hypoxic dormant CSCs and tumour-associated macrophage (TAM) polarization of bone marrow-derived monocytes (BMDMs).26. Trp53 is a key regulator of radiation responses in ECs, and tansforming growth factor-β (TGFβ)-related signalling potentially is a key regulator of EndMT28. Thus, we explored the effects of small interfering RNA (siRNA)-mediated knockdown of Trp53 and Tgfbr2 on radiation-induced EndMT in human umbilical vein ECs (HUVECs). At 48 h post irradiation (hpi), Trp53 silencing in HUVECs markedly inhibited irradiation-induced messenger RNA (mRNA) expression of Snail1, Snail2, and Zeb2, which encode transcription factors implicated in EndMT29, compared to control siRNA-treated cells, whereas Tgfbr2 knockdown increased their expression , while reversing irradiation inhibited CD31 levels  and vessels were more dilated than in non-irradiated tumours . The changes in tumour size are shown in Supplementary Fig. Trp53-knockdown (EC-p53KD) and -knockout (EC-p53KO) mice . A syngeneic tumour model was established by implanting tumour cells (referred to as KP cells) isolated from a spontaneous lung adenocarcinoma in conditional KrasG12D;Trp53flox/flox mice30. Primary KP cells were implanted at passage 4 or less to maintain the cellular characteristics of spontaneous lung tumour. Trp53 mRNA–VE-cadherin+ cells were dominant in EC-p53KO, but not wild-type (WT) tumours, indicating that p53 was successfully knocked out in tumour ECs of EC-p53KO mice  than in non-irradiated WT (day 15), but not EC-p53KO mice   at 7 dpi in tumours of EC-tdTomato, but not EC-tdTomato-p53KO mice  cells in WT tumours were less TUNEL– or γH2AX– at 3 dpi, but highly Ki67+ (indicative of cell proliferation) during tumour regrowth (23 dpi), in contrast to SMA− ECs in EC-p53KO mice  mice . Supplementary Figure Tgfbr2 knockdown. Post-irradiation p-SMAD2/3 levels of CD31+ vessels were significantly higher in EC-TGFβR2KD than in WT mice -labelled BMDCs  during tumour regrowth after radiotherapy -overexpressing CT26 cells, we verified that mainly CD44v6 was increased in GFP+ cell membranes during tumour regression and regrowth when compared to non-irradiated tumours , in contrast to CD44v6+ cancer cells in non-severely hypoxic areas of irradiated WT tumours  lung tumour colonies (>100 μm tumour diameter) were CD44v6+ vs. 25% in non-irradiated mice  analysis. Various EndMT progression-associated molecules were upregulated at 48 hpi  and upregulated fibroblast-specific genes  in KP ECs isolated from WT, but not EC-p53KO mice  and hypoxic (1%/0.5% O2) conditions with/without OPN, in vitro. Hypoxia increased CD44v6 expression, with non-proliferative (Ki67–) KP cells surviving after irradiation  and proliferative CD44v6+ cells in EC-TGFβR2KD tumours at 21 dpi, and it induced similar effects in WT tumours +F4/80+ TAMs (M2-type) were highly accumulated in irradiated WT, but not EC-p53KO tumour vessels, compared to non-irradiated control vessels  in WT tumours  M2 subtype vs. macrophages cocultured with non-irradiated tumour ECs . When cocultured with irradiated WT, but not EC-p53KO tumour ECs showing EndMT, F4/80ECs Fig. . In contECs Fig. . Cocultuold Fig. .+ cells were highly recruited to both irradiated WT and EC-p53KO tumours  is highly expressed on irradiated ECs, which is inhibited by Trp53 deficiency Fig. . Indeed,urs Fig. . SDF-1+ urs Fig. . Becauseurs Fig. . These M-1+ Fig. .Fig. 6EC+ monocyte differentiation to M1/M2 TAMs, irradiated tumour ECs were cocultured for 48 h with macrophages differentiated from BMDMs by M-CSF. In accordance with the in vivo results, most F4/80+SDF-1+ cells were CD206+ (M2) after coculture with WT tumour ECs, but iNOS+ (M1) after coculture with EC-p53KO tumour ECs  when differentiated with cytokines interleukin-4 (IL-4) and IL-10 from M-CSF-treated BMDMs, whereas after coculture with EC-p53KO tumour ECs, iNOS+SDF-1+F4/80+ cells were flattened (like M1 macrophages) when differentiated with lipopolysaccharides (LPS) plus interferon-γ (IFN- γ) from M-CSF-treated BMDMs  of SDF-1+F4/80+ cells, which was inhibited by AMD3100 + CSCs and M2 macrophages in lung tissues of patients with lung cancer who received radiotherapy (Supplementary Table n = 10), which were compared to non-irradiated human lung tissues (n = 10) , although they were detected more in irradiated than in non-irradiated tissues  therapy or chemotherapy feature perivascular cells with significantly increased SMA expression38. Thus, radiotherapy with anti-VEGF or anti-angiogenic therapy may be more efficient when combined with a strategy for inhibiting tumour EndMT.The findings of this study support a model for TRP53-regulated radiation-induced EndMT and tumour vasculature Fig. . EndMT i8. In our study, during early tumour regrowth, BMDCs recruited to EndMT vasculature after irradiation colocalized with SMA+ cells, but not with ECs. We hypothesize that the recruited BMDCs support SMA+ vessel maturation, as the origin of tumour-vascular pericytes was traced to mesenchymal progenitor cells or BMDCs39. A single, high dose (20 Gy) of non-curative radiation used to examine vasculature development during the initial-response, early-regrowth, and late-regrowth tumour phases was insufficient to eradicate all intratumour and peripheral tumour vessels or angiogenic vessels surrounding the tumours. In contrast, tumour EndMT was rarely detected in recurrent KP tumours after radiotherapy with 2 times 25 Gy  CSCs may become awakened by angiocrine factors (such as OPN) secreted during tumour EndMT-related revascularization around hypoxic regions. Furthermore, we consider CD44v6+ CSCs more important than other CSCs in radiotherapy, as CD44v6+ populations were induced by radiation and might be mainly responsible for post-radiotherapy tumour regrowth and metastasis.We found that EC-p53KO tumours regulated OPN, an angiocrine factor acting on radioresistant CSCs, and EC receptors, which in turn controlled TAMs, in the radioresistant tumour microenvironment, supporting the observed role of tumour angiocrine factors in the crosstalk between CSCs and tumour vessels3. High-dose radiation induces antigen production via tumour cell death, tumour-specific immunity, and tumour-vascular damage49. Radiation can attract immunosuppressive cells to the tumour microenvironment17. TAM modulation is considered important in cancer immunotherapy because of the tumour-promoting effects of enhancing an immunosuppressive microenvironment and tumour angiogenesis51. Klug et al.52 reported that low-dose irradiation stimulates immunostimulatory macrophages, enabling cytotoxic T cells to infiltrate tumours, thus boosting tumour immunity. In the current study, we discovered that tumour vascularization via EndMT enhanced TAM polarization toward M2-like macrophages and proliferation after radiotherapy. M0 macrophages differentiated from BMDMs prominently showed Arg1+ M2 polarization in coculture with radiation-EndMT-derived tumour ECs. However, BMDMs mainly polarized to the iNOS+ M1 subtype during coculture with ECs from EC-p53KO mice, in which radiation-induced EndMT was inhibited. Accordingly, in vivo, at 1 dpi, tumours recruited monocytes and F4/80+ macrophages. At 7 dpi, M2 macrophages were mainly seen in WT tumours, while M1 macrophages were predominantly observed in irradiated EC-p53KO tumours, indicating that the tumour-vascular niche regulates TAM polarization after radiotherapy. Detailed effects of radiation-induced EndMT on other immune cells, including cytotoxic T and antigen-presenting cells, remain to be explored.Irradiation seems to modulate tumour immunogenicity in conventional radiotherapy and stereotactic ablative radiotherapy50. Interestingly, strongly SDF-1+ monocytes and subsequently SDF-1+CD206+ M2 macrophages were recruited to KP tumours after radiotherapy. Furthermore, EndMT tumour-derived ECs highly expressing CXCR4 induced M2 polarization and proliferation of SDF-1+ macrophages, which was inhibited by Trp53 silencing. AMD3100 significantly inhibited M2 SDF-1+ macrophage polarization in coculture with irradiated tumour ECs. Relevant to our findings with SDF-1+ macrophages, SDF-1 produced in monocytes contributes to macrophage differentiation36. In accordance with previous findings53, SDF-1+ tumour cells were observed after radiation, but in this study, SDF-1 expression was higher in monocytes than in tumour stromal cells; this discrepancy may reflect a requirement for SDF-1 cleavage for cellular activity, as monocytic SDF-1 was not strongly detected with antibodies targeting the C terminus of SDF-1. We are currently studying SDF-1 cleavage in relation to monocyte differentiation.High SDF-1/CXCL12 expression in tumours aids in the capture of CXCR4-expressing monocytes11. In this study, targeting EndMT more efficiently inhibited tumour regrowth after a single high dose of radiation than after fractionated radiotherapy. In fractionated radiotherapy, tumour EndMT was significantly inhibited by EC-p53KO; however, CD44v6+ CSC proliferation and tumour growth were not inhibited. Nevertheless, we cautiously suggest that in fractionated radiotherapy, targeting EndMT may enhance therapeutic efficacy when combined with inhibition of CSC proliferation.The effect of radiation-induced tumour-vascular damage on TAM polarization, which was increased by EndMT, suggests that high-dose radiation (~10 Gy) can evoke stronger tumour immune responses than low-dose radiation, because it indirectly causes tumour cell death via vascular damage+CD44v6+ CSCs and SDF-1+CD206+ macrophages, even though the patients had received fractioned radiotherapy, indicating that radiation-induced CD44v6+ tumour cells and SDF-1+ M2 macrophages might be targeted for overcoming tumour radioresistance.In accordance with our findings in vivo, tumour EndMT was prominently seen in lung cancer tissues of patients who received irradiation, with large populations of OPNIn conclusion, this study provides evidence that targeting radiation-induced EndMT is a new, promising strategy for enhancing tumour radiosensitivity by inhibiting radiation-induced abnormal tumour vasculature. Specifically, targeting vascular EndMT may be effective in regulating radioresistant CSC proliferation and maximizing the anti-tumour immunity of radiotherapy.54. Specific pathogen-free C57BL/6 Tie2-Cre, Trp53flox/flox, Tgfbr2flox/flox, and LSL-KrasG12D mice were purchased from the Jackson Laboratory. Tie2-GFP mice were a kind gift from Dr. Gou Young Koh (Korea Advanced Institute of Science and Technology). Male Tie2-Cre mice were crossed with female Trp53flox/flox, Tgfbr2flox/flox, or Trp53flox/floxTgfbr2flox/flox (generated by crossing Trp53flox/flox and Tgfbr2flox/flox) mice to generate Tie2-Cre;Trp53flox/+, Tie2-Cre;Trp53flox/flox, Tie2-Cre;Tgfbr2flox/+, Tie2-Cre;Trp53flox/+Tgfbr2flox/+, or Tie2-Cre;Trp53flox/floxTgfbr2flox/+ mice. Tie2-Cre;Trp53+/+, Tie2-Cre;Tgfbr2+/+, Tie2-Cre;Trp53+/+Tgfbr2+/+, or Tie2-Cre–/– littermates were used as controls. All animal experimental data shown are representative of three independent experiments. All experiments were conducted with 6–8-week-old mice. The mice had access to a standard diet and water ad libitum. All mice were anaesthetized with a combination of anaesthetics before being killed. For in vivo proliferation assays, mice received an intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdU) (2 mg/mouse) (Sigma-Aldrich) 4 h before tumour harvesting. BrdU+ cells were detected using an anti-BrdU mouse monoclonal antibody .All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Radiological & Medical Sciences and are reported in accordance with the ARRIVE  guidelinesn = 7; Origene tissues, n = 3) who received radiotherapy and 10 patients  who did not receive radiotherapy are shown in Supplementary Table The analysis of lung cancer patient tissues was approved by the ethics committee of Severance Hospital, Yonsei University (Korea). In addition, a lung cancer tissue with information related to radiotherapy was purchased from Origene (cat. no. CT565899). Clinicopathological characteristics of 10 patients  were injected subcutaneously into the right thigh of 8-week-old male BALB/c mice. When the tumour volume reached 150–200 mm3, tumours or whole mice were irradiated with a sublethal dose (8 Gy). Bone marrow cells were harvested from the tibias and femurs of male BALB/c donor mice and were fluorescently labelled with CM-Dil dye , per the manufacturer’s instructions. CM-Dil-labelled cells (2 × 107) were suspended in 200 μl of Opti-MEM (Gibco) and intravenously injected into whole body-irradiated mice at 6 hpi. Tumours from control and transplanted mice were harvested at 7 dpi. KP tumour-bearing recipient mice were sub-lethally irradiated (9 Gy), and 9 h after irradiation, they received Tie2-GFP mouse-derived bone marrow cells (1 × 107 cells) intravenously and were allowed to recover for 3 weeks.CT26 cells .Tumour tissues were covered with optimal cutting temperature (OCT) compound  and snap-frozen on dry ice or fixed in 10% (v/v) neutral-buffered formalin, embedded in paraffin, and sectioned. The sections were deparaffinized and stained as previously described5), differentiated by treatment with murine M-CSF every other day for 7 days, were placed on the tumour ECs. The cocultures were harvested at 48 h. Tumour ECs and BMDMs cultured alone were used as controls.For coculture experiments, tumour ECs were plated in 6-well plates, with or without radiation. BMDMs (1.2 × 105) were injected subcutaneously into the right thighs of C57BL/6 mice. When the tumours reached 150–200 mm3, a single dose of radiation (20 Gy) was delivered using the X-RAD 320 platform (Precision X-ray). AMD3100 (Sigma-Aldrich) was dissolved in distilled water, diluted in phosphate-buffered saline, and administrated daily intraperitoneally (5 mg/kg) for 7 days.KP tumour cells (2 × 105) were injected subcutaneously into the right thighs of male BALB/c mice. KP tumour cells (2 × 105) were injected subcutaneously into the right thighs of Tie2-Cre;Trp53flox/+, Tie2-Cre;Trp53flox/flox, Tie2-Cre;Tgfbr2flox/+, Tie2-Cre;Trp53flox/+Tgfbr2flox/+, Tie2-Cre;Trp53flox/floxTgfbr2flox/+, Tie2-Cre;R26RtdTomato;Trp53flox/flox, and VE-cadherin-CreERT2;Trp53flox/flox mice and control littermates. Tumour volumes were determined according to the formula (L × W × H)/2, using the calliper-measured tumour length (L), width (W), and height (H). When tumour volumes reached 150–200 mm3, radiation was delivered, using the X-RAD 320 platform (Precision X-ray). For OPN blocking experiments, mice were intraperitoneally injected with an anti-mouse OPN neutralizing antibody  four times, every 2 days.CT26 cells  1 min prior to killing. For hypoxia assessment, pimonidazole (60 mg/kg) was administered intraperitoneally. After 45 min, the mice were killed and tumours were harvested, fixed, and immunostained using the Hypoxyprobe Plus Kit (Hypoxyprobe) per the manufacturer’s instructions. To evaluate tumour metastasis, metastatic nodules on lung surfaces observed by the naked eye were counted. To visualize tumour vessel leakage, mice were intravenously injected with fluorescein isothiocyanate–dextran  5 min before killing.At day 7, tumour samples were harvested and digested into single-cell suspensions. Cells were stained with macrophage-specific antibodies. The cells were fixed and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences) and stained with an anti-SDF1 antibody. Then, the cells were analysed on a FACSCalibur flow cytometer . Gating strategies for flow cytometry analysis are shown in Supplementary Figure 137Cs] source (Atomic Energy of Canada) at 3.81 Gy/min. To establish hypoxia, cells were incubated in a Forma 1025/1029 Anaerobic Chamber (Thermo Fisher Scientific) flushed with 0.5% or 1% O2 for severe or mild hypoxia, respectively. Cells were incubated with 10 μM EdU for 2 h, trypsinized, and surface-stained with an anti-CD44v6 antibody . The cells were labelled using the Click-iT Alexa Fluor 488 Assay Kit  and analysed on a FACSCalibur .For in vitro proliferation assays, KP cells were irradiated with gamma rays from a  source (Atomic Energy of Canada) at 3.81 Gy/min. For the analysis of secreted proteins, conditioned media were collected between days 6 and 7 of HPMEC culture after irradiation and assayed using Proteome Profiler Human Soluble Receptor Array, Hematopoietic Kit , according to the manufacturer’s instructions. For the analysis of cellular proteins, immunocytochemistry was performed as previously described26. OPN released by HUVECs into the culture medium was measured using a human OPN Quantikine ELISA Kit (R&D Systems), following the manufacturer’s instructions.HUVECs, human pulmonary microvascular endothelial cells (HPMECs), and human pericytes were obtained from PromoCell  and were cultured in Endothelial Cell Growth Medium 2, Endothelial Cell Growth Medium MV2, and Pericyte Growth Medium, respectively (Promocell) under 5% CO55. Fragments-per-kilobase-of-transcript values were calculated with Cufflinks55. Genes with a maximal log2-transformed read count <5 across samples were excluded from the analysis. To identify differentially expressed genes, we compared the direction of fold changes (>1.2) against irradiation (IR) alone. Reverse-regulated genes (>1.2-fold) between TGFBR2 knockdown+IR and p53 knockdown (with and without TGFBR2 knockdown)+IR compared with IR alone were selected. To analyse angiocrine factors and phenotypic changes in endothelial cells, matrisome gene lists and surfactome gene lists were used. The matrisome comprises core matrisome-related proteins, including extracellular matrix (ECM) glycoproteins, collagens, and proteoglycans, as well as ECM-associated proteins, including ECM-affiliated proteins, ECM regulators, and secreted factors56. The surfactome represents proteins at the plasma membrane, including G protein-coupled receptors, receptor tyrosine kinases, and integrins57. Among the differentially expressed genes, matrisome and surfactome genes were displayed in R using the ComplexHeatmap package58 and were used as input for the GeneMANIA 3.4.1 plugin59 within Cytoscape 3.4.060 for functional enrichment analysis. RNA-seq data generated in this study are available on Gene Expression Omnibus Total RNA was isolated from HUVECs, and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA-seq libraries were constructed using the SENSE mRNA-Seq Library Prep Kit (Lexogen), according to the manufacturer’s instructions, and were sequenced as 100 bp paired-end runs on the HiSeq 2000 platform (Illumina). RNA-seq reads were mapped to UCSC hp19 using TopHat softwareGAPDH. Primers are listed in Supplementary Table RNA was isolated using TRI reagent , and 1 μg of RNA was used to synthesize complementary DNA with an Omniscript RT Kit . PCR was conducted in triplicate on the CFX96 TM Real-Time system , using qPCR SYBR Green master mix (Invitrogen). For each sample, target gene expression was normalized against the geometric mean of the reference gene t-test (Figs. p value < 0.05 was regarded significant. Experimenters were blinded to group assignments and outcome assessments.Student’s st Figs. , 7a–d anSupplementary InformationPeer Review File


MRI using fluorinated probes enables the achievement of a specific signal with high contrast in MRI images. However, to ensure sufficient sensitivity of 19F MRI, fluorine probes with a high content of chemically equivalent fluorine atoms are required. The majority of 19F MRI agents are perfluorocarbon emulsions, which have a broad range of applications in molecular imaging, although the content of fluorine atoms in these molecules is limited. In this review, we focus mainly on polymer probes that allow higher fluorine content and represent versatile platforms with properties tailorable to a plethora of biomedical in vivo applications. We discuss the chemical development, up to the first imaging applications, of these promising fluorine probes, including injectable polymers that form depots that are intended for possible use in cancer therapy.Over the last few years, the development and relevance of  For this reason, fluorine isotopes are visualized with high specificity, allowing for so-called “hot spot” imaging where the 19F MR signal is overlaid on a proton reference MR image. Moreover, the fluorine isotope 19F resonates at a Larmor frequency close to that of the proton 1H (94% of 1H); therefore, the hardware used for 1H MR imaging, with minor modifications, can also be used for 19F MRI. Currently, many clinical and experimental scanners have become available for hardware modifications and the use of appropriate radiofrequency (RF) coils. Usually, the RF coils intended for 19F MR are constructed to be dual-tuned for both 1H/19F MRI. The 19F MR signal is proportional to the number of 19F nuclei, which allows absolute quantification of the 19F content.Magnetic resonance imaging (MRI) using fluorinated probes  or exogenous labeling of cells. Currently, there is a wide range of available fluorine probes for experimental and even clinical applications summarized in many excellent reviews  (PDFEA)  segments played an important role in the accumulation of the probe in biological tissue. The polymer showed resistance to rapid uptake by macrophages and thus longer circulation times in the blood. This effect is also seen in other studies  (PDMAEMA) and OFPMA, which self-assemble into micelles in either an acidic aqueous solution or in a dimethylformamide/water mixture (both at pH 3). The size  of the micelles depends on the length of the DMAEMA segment and varies with pH. The authors reported various sizes (50–200 nm) and structures for the synthesized probes ranging from micelles to fibers depending on the length of the protonated PDMAEMA chains. Fluorine MR spectra showed multiple 19F peaks for the polymer.In 2007, the group of Mao et al.  presenteN,N-diethylamino)ethyl methacrylate] (PDEA) were synthesized by the Zhang  (PHPMA) or poly(2-methyl-2-oxazoline) (PMeOx) as the hydrophilic block and PDFEA as the fluorinated thermoresponsive block exhibited thermal responsivity, being molecularly soluble at room temperature and forming well-defined nanogels at body temperature  for visualization by 19F MRI using standard spin-echo sequences. The presence of both polymers (PMeOx-PDFEA and PHPMA-PDFEA) was observed by 19F MRI after injection either into the muscle of mice or subcutaneously (Fig. Self-assembled biocompatible polymer nanogels consisting of hydrophilic-thermoresponsive diblock copolymers containing either poly[ure Fig.  39]. Th. ThN-(2-19F atoms, the localization, size and biodegradation of the implants can be monitored by 19F MRI with high sensitivity due to the high local 19F concentration at the injection site. In our recent study [19F MRI after intramuscular and subcutaneous administration in rats (Fig. Fluorinated thermoresponsive polymers can also be implemented for sustained release of drugs from implants, which are created upon injection into the body . Typicalnt study , we usedats Fig. . The dep19F MRI using fluorine-containing agents represents a specific imaging method with applications in both experimental and clinical medicine due to the negligible 19F MR signals from the body. To achieve sufficient sensitivity of 19F MRI, novel fluorine-containing probes are needed. Polymer-based probes are a very promising and versatile platform with properties tailorable to a plethora of in vivo biomedical applications. In particular, stimuli-responsive supramolecular polymer nanostructures allowing for functional imaging in response to changes in temperature, pH, reactive oxygen species and other physiological stimuli hold promise for noninvasive functional diagnostics of pathological tissues in the future. Targeted cell labeling for long-term tracking of transplanted cells by 19F MRI is another highly promising direction for such 19F agents.19F MRI polymer probes in practice, especially in clinics, several properties of these probes must be fulfilled. Among them are a high content of fluorine atoms that are chemically equivalent so that all of them fit into the chemical shift window of interest, an efficient mobility and limited association of the fluorine segments resulting in suitable relaxation times, an adequate solubility in water, an easily modifiable structure for targeting, reliable pharmacokinetic/pharmacodynamic properties including biodegradability, clearance of the probe after the system fulfills its task, and the capability of functional imaging if needed in the particular application. Another important challenge that must be solved is the biodegradability of the imaging agents so that the system may be fully eliminated from the organism after fulfilling its task.To employ the


Specially, the calculated results demonstrate that Probe1 possesses a higher energy transfer efficiency and a larger two-photon absorption cross-section than Probe2, indicating it to be a preferable pH fluorescent probe. Therefore, the influence of connection between the donor and the acceptor on the sensing performances of the probe is demonstrated. Our results help to understand the experimental observations and provide a theoretical basis to synthesize efficient two-photon fluorescent probes for monitoring pH changes.The design and synthesis of fluorescent probes for monitoring pH values inside living cells have attracted great attention, due to the important role pH plays in many biological processes. In this study, the optical properties of two different two-photon fluorescent probes for pH are studied. The ratiometric sensing of the probes are theoretically illustrated. Meanwhile, the recognitional mechanisms of the probes are investigated, which shows the energy transfer process when react with H Intracellular pH value plays a pivotal role in biological processes, such as the growth of the cell, transportation of the ions, shrinkage of muscle, and activity of the enzyme ,2. AbnorThe value of pH can be assessed by various techniques, including nuclear magnetic resonance, permeable microelectrodes, absorbance spectroscopy, and fluorescence microscopy. Thereinto, fluorescence microscopy with pH-sensitive probes has become an indispensable tool, due to its high sensitivity and selectivity, simple operation, and non-destructive use in living cells ,9. In thUp to now, a variety of two-photon fluorescent probes have been developed for measuring pH. For instance, Kim et al. reported a new family of small molecule and ratiometric two-photon probes derived from benzimidazole for monitoring acidic pH values based on the intramolecular charge transfer (ICT) mechanism . Besides+. Special attention has been paid to analyzing the recognitional mechanism of the probes using a molecular orbital diagram. Importantly, a feasible approach has been used to predict the energy transfer rate between the energy donor and acceptor of the probe. Our theoretical investigations can provide helpful information for rationally designing fluorescent probes with high efficiency.Many FRET/TBET-based fluorescent probes have been put forward for the ratiometric detection of pH so far ,21,22. VIn this work, geometrical structures of all the studied molecules are fully optimized by using the density functional theory with the 6-31G basis set, and the B3LYP hybrid functional . Frequenijω denotes the energy difference between the states i and j, αμ is the electric dipole moment operator, and the summation is performed over the axes The transition probability of one-photon absorption and emission can be described by the oscillator strengthMoreover, the DALTON2013 program  is emploμα(β) denotes the dipole moment operator in the direction α, βє; ω is the energy of the incident laser beam and equal to half of the excitation energy of the final state j; and the summation covers all the intermediate states. The TPA cross-section of a molecule is given by orientational averaging over the TPA probability:From the sum-over-state formulas, the resonant two-photon absorption matrix element can be expressed as (2)Sαβ=∑F, G, and H are related to the polarization of the excitation laser pulse. For linearly polarized light, the values of F, G, and H are 2, 2, and 2; and for the circular case, the values are—2, 3, and 3, respectively. The summation covers the molecular x, y, and z axes, namely, α, βє.Here, the coefficients The TPA cross-section, directly comparable with experiment, is then defined as (4)σTPA=a0, α, and c are the Bohr radius, the fine structure constant, and the speed of the light, respectively; ħω is the incident photon energy; g(ω) provides the spectral line profile; and the lifetime broadening of the final state Γ is assumed to be a typical value of 0.1 eV.Here, +, the ring-closed rhodamine in the probes are induced to be ring-opened forms, as shown in Probe1+H+ and Probe2+H+. The optimized ground state geometries of the molecules in H2O show that the donor and acceptor moieties of the probes, in the absence and presence of H+, are coplanar, and there is a large tortuosity between them .The OPA process is closely related to the fluorescence of the molecules, thus, it is necessary to analyze the molecular OPA properties. The absorption spectra of the studied molecules in H0 to the second excited state S2. For Probe1+H+  and the rhodamine B (the acceptor). Due to the fact that the energy transfer process actually means the acceptor part is excited by absorbing the fluorescence of donor, we thus calculate the emission of the donor moiety and the absorption of the acceptor moiety. In this paper, the short axis of the xanthene structure of rhodamine, the long axis of the xanthene, and the direction perpendicular to the plane of xanthene are set to be the 2+H+ see . On basix-axis direction, both for Probe1+H+ and Probe2+H+. Although the total distances between the donor and acceptor are almost the same for the two molecules, the energy transfer rate of Probe1+H+ is about 2.7 times larger than that of Probe2+H+, indicating that Probe1 can be a preferable probe with higher efficiency.As is known, the energy transfer rate depends crucially on the distance vector between the donor and acceptor of the molecule . Data in+. Importantly, the naphthalimide derivative is reported as a good two-photon fluorophore. Hence, the TPA properties of the studied molecules are theoretically discussed. In the range of 700–900 nm, the two-photon excitation energy, the corresponding TPA wavelength, and the TPA cross-section are summarized in σTPA < 50 GM). In the presence of H+, the maximum TPA cross-sections of Probe1+H+ and Probe2+H+ exhibit great enhancement from 47 to 300 GM, and from 27 to 117 GM, respectively, which is contributed to by the open-ring rhodamine. Note that the maximum TPA cross-section of Probe1 is much larger than that of Probe2, both in the absence and presence of H+. Due to the probes possessing similar donor and acceptor, but different connections, it can be concluded that the connection between the donor and acceptor plays a dominant role in the TPA performance. As a consequence, Probe1 is proven to be a preferable candidate as the two-photon fluorescent probe for H+ in comparison with Probe2.The analyses on the fluorescence and recognitional mechanism of the probes have demonstrated that Probe1 and Probe2 can effectively identify H+ is present, the energy transfer process will occur in Probe1+H+, and the fluorescence from the acceptor can be observed. In this case, the detection of H+ is efficiently achieved, with weaker photodamage and photobleaching.For Probe1, using a laser with 826 nm as the excitation source, the probe is excited and the fluorescence emitted from the donor part can be observed on account of no energy transfer. When H+, indicating the probes can serve as efficient fluorescent probes for ratiometric pH measurements. Molecular orbital diagrams are utilized to elucidate the sensing mechanisms of the probes, where the TBET and FRET processes have been specifically modeled. Importantly, the energy transfer rates of the energy transfer-based fluorescent probes are compared based on the same theory level, and the results reveal that Probe1 has a higher energy transfer rate, suggesting that Probe1 can be a promising TBET-based fluorescent sensor compared with Probe2. In addition, two-photon absorption cross-section is largely enhanced when the probes react with H+. It shows that both probes are efficient two-photon fluorescent probes. Specially, the two-photon cross-section of Probe1 is larger both in the absence and in the presence of H+, which confirms Probe1 to be a better H+ chemosensor. Further, the effect of the connection between the donor and the acceptor of the probe is demonstrated. Our theoretical investigations revealed the underlying mechanisms that satisfactorily explained the experimental results, providing efficient information on designing more two-photon fluorescent probes.In this work, the optical properties, recognitional mechanisms, and energy transfer rate of two fluorescent chemosensors for pH are theoretically investigated. The calculated results show that the photoabsorption and photoemission of the probes are significantly changed with the addition of H


To investigate the mechanism of the protective effect of Zishenwan on pyelonephritis rats.κB pathway were detected by Western blot in renal and urinary tract mucosa. The protective effect and influence on TLR4-NFκB pathway of Zishenwan were studied. In the rat model of pyelonephritis, protective effects of Zishenwan, the content of secretory immunoglobulin A (SIg A), and interleukins were detected by ELISA. The expressions of TLR4-NFκB pathway. Serum containing Zishenwan can significantly decrease LPS-induced expression of TLR4, MyD88, and NFκB in vitro. And the inhibition of TLR4 signal by Zishenwan related to the degradation of TRAF3 and TRAF6. Zishenwan protected rats from pyelonephritis which related to the increase of SIgA, the regulation of interleukins, and the inhibition of TLR4-NF Zishenwan protected rats from urinary tract infection by clearance of bacteria and decrease of tissue damage. 20S proteasomes mediated the degradation of TRAF3 which is important to the decrease of tissue damage from Zishenwan. Urinary tract infection (UTI) is a common infectious disease in the general population . Accordi+T cell number and ratio of CD4+/ CD8+ T cells. So Zishenwan has protective effects other than the direct antimicrobial activity.In traditional Chinese medical science, recurrent UTI belongs to the category of “Lao Lin" and “deficiency of kidney and bladder heat” is the pathogenesis of recurrent UTI. “Zishenwan" comes from the “LanShiMiCang”, and it is an effective drug treating “Lin syndromes” for a long time. Some components in Zishenwan have antimicrobial activities and it might be an important factor in the treatment of recurrent UTI –8. But aMucosal immunology is an important mechanism in protecting infection , 10. It  Escherichia coli (UPEC) can be identified by TLR4 and induce cytokine production through MyD88-NFκB pathway in renal tubular epithelial cells [TLR4 signaling pathway plays important roles in the development of immune diseases in clinic . During al cells , 17. Meaal cells . TherefoIn our experiments, we observed beneficial effects of Zishenwan in chronic pyelonephritis rats and these beneficial effects might come from the secretion of SIgA in the urethral mucus and inhibition of TLR4 signal in rat bladder smooth muscle cell. Our results showed a novel mechanism of Zishenwan in the treatment of recurrent urinary tract infection.Male SD rats (180-220 g) were supplied by JOINN Laboratories (Suzhou); license number is scxk (苏) 2013-0003. All surgical procedures and care administered to the animals were approved by the institutional ethic committee, and this study also complied with the criteria in Guide for the Care and Use of Laboratory Animals of Suzhou Hospital of Traditional Chinese Medicine. Escherichia coli (E.coli) O111B4  was injected into the bladder and the thread on penis was released after 24 hours. The model of chronic pyelonephritis was evaluated after 6 weeks.Rat model of chronic pyelonephritis was established following the protocol of Goluszko et al. After fasted for 24 hours, penises of rats were bound with thin thread after fasted for 18 hours. Then 0.5ml ofRats which were induced with chronic pyelonephritis successfully were divided into 3 groups randomly; model group (10ml pure water/kg), Zishenwan group (27.7g crude drug/kg), and Levofloxacin group (0.23g/kg). Untreated rats were used as control group and were administrated with pure water (10ml/kg). 8 rats per group and they were administrated for 30 days. After the last administration, rats were fasted for 18 hours and anaesthetized with pentobarbital sodium. Then, urine and kidneys were collected.μm-thick slices, and then examined via light microscopy  for hematoxylin-eosin (HE) immunohistochemistry.Tissues from mice were fixed in 4% paraformaldehyde, dehydrated for 12 hours, embedded in paraffin wax, cut into 3Rat bladder smooth muscle cell is a gift from China Pharmaceutical University and cultured in complete medium .15 male SD rats were randomly divided into 3 groups: normal control serum group, low dose serum group, and high dose serum group (n=5). Rats from low dose serum group were administrated with Zishenwan (5.54g crude drug /kg) daily and rats from high dose group were administrated with Zishenwan (27.7g crude drug /kg) daily. Rats from normal serum group were administrated with saline: the rat body surface area ratio is converted into the equivalent to clinical dose, respectively, to 1, 5 times the amount of medication, and normal serum group were given equal volume of saline, with continuous administration for 7 days. Serum was inactivated at 56°C for 30 min and sterilized by filtration; then it was stored at -80°C. The drug serum and serum free DMEM culture medium were allocated according to the ratio of 1:9, and the culture medium containing 10% serum was obtained.Immunoglobulin A (IgA) in the urine was measured using Rat Immunoglobulin AELISA Kit . Collected urines were centrifuged at 5,000g for 15 min to remove insoluble matters, and the supernatant was used for ELISA assay. The level of SIgA was assessed by ELISA according to the protocol of the supplier.Interleukins in the urethral mucus were measured using ELISA kit . The urethral mucus was separated carefully and weighted. Then, the urethral mucus was powdered into liquid nitrogen and mixed with PBS (50mg/ml). The mixture was violently shocked at 4°C and centrifuged at 17,500g for 15 min to remove insoluble matters. Levels of IL-4, IL-6, and IL-10 in supernatant were assessed by ELISA using monoclonal antibodies and the procedure recommended by the supplier.μL binding buffer containing 5 μL annexin V (10 μg/mL) and 10 μL PI (20 μg/mL) for 30 min. Then samples were immediately analyzed using flow cytometry.The apoptotic cells were quantified using the annexin V and PI double staining kit (BD Biosciences). Briefly, cells were collected, washed with PBS, and resuspended in 200 β-Actin , TLR4 , MyD88 , NFκB/p65 , p-IκBα (Ser32/Ser36) , IκBα , TRAF3 , and TRAF6  at 4°C overnight, the blots were washed in PBS containing 0.1% Tween and incubated with secondary antibodies  for 1h at room temperature. Blots were then washed four times with PBS containing 0.1% Tween. Immunolabeling was detected by enhanced chemiluminescence.Rat bladder smooth muscle cells and homogenate treated samples of tissues were lysed in RIPA  and the protein concentration was determined by a Bradford kit . The proteins were treated with SDS sample buffer at 100°C for 10 min and resolved by SDS-polyacrylamide gel electrophoresis (10% acrylamide) and transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with PBS containing 3% bovine serum albumin (BSA) for 1h at room temperature. Following incubation with the primary antibodies specific for  p<0.05.Results were expressed as mean ± standard deviation (SD). The data were analyzed by one-way ANOVA analysis of variance followed by Dunnett's test. Statistical differences were considered statistically significant whenFrom the results of HE stain, infiltration of a large number of inflammatory cell, interstitial fibrous tissue hyperplasia and fibrosis, telangiectasia, interstitial fibrous proliferation, telangiectasia, interstitial hyperemia and edema, necrosis, and loss of epithelial cells of renal pelvis mucosa were observed in model group . The admComponents from Zishenwan were reported to inhibit the growth of bacteria, such as berberine and cinnamaldehyde, but beneficial effects of Zishenwan in pathological changes were much more significant. So we hypothesized that factors other than direct antibiotic activity should contribute to the treatment effect of Zishenwan. At first, we detected the secretory immunoglobulin A (sIgA), an important antibody relating to mucosal defense, and we found the concentration of sIgA increased in model group, Levofloxacin group, and Zishenwan group  to induce apoptosis of rat bladder smooth muscle cell and observed protective effects from Zishenwan serum ( in vitro (Figures From previous results, Zishenwan decreased the expression of TLR4 more effectively than Levofloxacin, and TLR4 expressing in normal cells can induce damage directly. So we speculated that Zishenwan inhibited TLR4 pathway to protect kidney beside the inhibition of infection.an serum . In the κB was not only the downstream factors of TLR4 but also the activators of transcription. The activation of NFκB relates to the concentration IκBα. LPS significantly increased phosphorylation of IκBα and decreased IκBα (κBα and induced the increase of IκBα (κBα and TRAF3 was abolished by the administration of MG132, a proteasome inhibitor (κBα-mediated TLR4-NFκB signal.In western-blot, TLR4 and its downstream molecular MyD88 were significantly increased after the administration of LPS and decreased by Zishenwan serum ( of IκBα , p<0.05. of IκBα , p<0.05.nhibitor , p<0.05.nhibitor , p<0.01, E. coli [, Anemarrhena asphodeloides and Cinnamomum cassia. Berberine in golden cypress has been reported to have inhibitory effects on dysentery bacillus, Staphylococcus aureus, and other pathogenic bacteria [ Anemarrhena asphodeloides has been reported to have inhibitory effects on E. coli, Proteus, and other bacteria [ Cinnamon oil and its main component cinnamaldehyde are reported to inhibit growth of Gram negative bacteria and Gram positive bacteria [Pyelonephritis is a kidney disease caused by urinary system infection causing serious complications and a few recurrent or delayed renal failures , 19. At  E. coli , 19. Ant E. coli . Another E. coli , 22. Tra E. coli , 5. Zishbacteria . Anemarrbacteria . Cinnamobacteria , 23. So,But Zishenwan should nourish kidney, clear heat, and clear Qi according to the theory of traditional Chinese medical science which seems different to the antibacterial effect. So we hypothesized that Zishenwan might increase protective effects of immune system and inhibit the tissue damage. Increased secretion of IgA which plays great important role on the resistance to adhering of pathogens to the urinary tract was proved after the administration of Zishenwan. The result boosted our confidence that Zishenwan has other mechanisms benefiting the pyelonephritis patients. From the results, tissue damage was significantly decreased and the effect was much better than the treatment of Levofloxacin. So the direct protection of tissue other than antimicrobials effect should exist in the treatment of Zishenwan.κB which controls transcription of interleukins were activated in urethral mucus of model rats. After the treatment of Zishenwan inhibited the activations of TLR4 pathway and NFκB were inhibited, and interleukins related to tissue damage were also decreased. This result was also proved in vitro. So Zishenwan can decrease Gram negative bacteria induced tissue damage by immune regulation.Gram negative bacteria are major pathogenic microorganisms and they are rich in LPS, a key ingredient inducing tissue damage. TLR4, an important receptor of LPS, was increased in urethral mucus of model rats. And the activation of TLR4 increases immune injury and induces direct tissue damage , 17. MeaFrom our results, Zishenwan can protect kidney from damage induced by LPS by the inhibition of TLR4 related pathway and it is other than the direct antibacterial activity. This effect is more consistent with theory of traditional Chinese medical science and it might connect the traditional Chinese medical science with modern biological theory.