Profile analysis and functional modeling identify circular RNAs in nonalcoholic fatty liver disease as regulators of hepatic lipid metabolism.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, associated with an outcome of hepatic fibrosis/cirrhosis and hepatocellular carcinoma. However, limited exploration of the underlying mechanisms hinders its prevention and treatment. To investigate the mechanisms of epigenetic regulation in NAFLD, the expression profile of circular RNA (circRNA) of rodents in which NAFLD was induced by a high-fat, high-cholesterol (HFHC) diet was studied. Modeling of the circRNA-microRNA (miRNA) -mRNA regulatory network revealed the functional characteristics of NAFLD-specific circRNAs. The targets and effects in the liver of such NAFLD-specific circRNAs were further assessed. Our results uncovered that the downregulation of 28 annotated circRNAs characterizes HFHC diet-induced NAFLD. Among the downregulated circRNAs, long intergenic non-protein coding RNA, P53 induced transcript (LNCPINT) -derived circRNAs (circ_0001452, circ_0001453, and circ_0001454) targeted both miR-466i-3p and miR-669c-3p. Their deficiency in NAFLD abrogated the circRNA-based inhibitory effect on both miRNAs, which further inactivated the AMPK signaling pathway via AMPK-α1 suppression. Inhibition of the AMPK signaling pathway promotes hepatic steatosis, depending on the transcriptional and translational upregulation of lipogenic genes, such as those encoding sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FASN) in hepatocytes. The levels of LNCPINT-derived circRNAs displayed a negative association with hepatic triglyceride (TG) concentration. These findings suggest that loss of LNCPINT-derived circRNAs may underlie NAFLD via miR-466i-3p- and miR-669c-3p-dependent inactivation of the AMPK signaling pathway.

Regulation of the macrophage-hepatic stellate cell interaction by targeting macrophage peroxisome proliferator-activated receptor gamma to prevent non-alcoholic steatohepatitis progression in mice.

BACKGROUND & AIMS: Macrophages display remarkable plasticity and can interact with surrounding cells to affect hepatic immunity and tissue remodelling during the progression of liver diseases. Peroxisome proliferator-activated receptor gamma (PPARγ) plays a critical role in macrophage maturation, polarization and metabolism. In this study, we investigated the role of PPARγ in macrophage-hepatic stellate cell (HSC) interaction during non-alcoholic steatohepatitis (NASH) development. METHODS: Wild-type, Ppargfl/fl and PpargΔLyz2 mice were fed a methionine- and choline-deficient (MCD) diet to induce NASH. Depletion of macrophages was performed using an injection of gadolinium chloride intraperitoneally. PPARγ-overexpressing or PPARγ-knockout macrophages were stimulated with saturated fatty acid (SFA) and cocultured with HSCs in a conditioned medium or the transwell coculture system. RESULTS: Depletion of macrophages inhibited HSC activation and ameliorated NASH progression in MCD diet-fed mice. Coculturing HSCs with macrophages or culturing HSCs in a macrophage-conditioned medium-facilitated HSC activation, and this effect was magnified when macrophages were metabolically activated by SFA. Moreover, the absence of PPARγ in macrophages enhanced metabolic activation, promoting the migration and activation of HSCs through IL-1ß and CCL2. In contrast, overexpression of PPARγ in macrophages obtained the opposite effects. In vivo, macrophage-specific PPARγ knockout affected the phenotype of hepatic macrophages and HSCs, involving the MAPK and NLRP3/caspase-1/IL-1ß signalling pathways. Infiltrating hepatic monocyte-derived macrophages became the predominant macrophages in NASH liver, especially in PpargΔLyz2 mice, paralleling with aggravated inflammation and fibrosis. CONCLUSIONS: Regulating macrophage PPARγ affected the metabolic activation of macrophages and their interaction with HSCs. Macrophage-specific PPARγ may be an attractive therapeutic target for protecting against NASH-associated inflammation and fibrosis.

Regulation of PPAR-γ activity in lipid-laden hepatocytes affects macrophage polarization and inflammation in nonalcoholic fatty liver disease.

BACKGROUND: Lipid metabolism disorder and inflammatory-immune activation are vital triggers in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Various studies have shown that PPAR-γ exerts potent anti-inflammatory and immunomodulatory properties. However, little is known about the regulation of PPAR-γ activity in modulating cell crosstalk in NAFLD. AIM: To investigate whether the regulation of PPAR-γ activity in lipid-laden hepatocytes affects macrophage polarization and inflammation. METHODS: Primary hepatocytes were isolated from wild-type C57BL6/J mice or hepatocyte-specific PPAR-γ knockout mice and incubated with free fatty acids (FFAs). Macrophages were incubated with conditioned medium (CM) from lipid-laden hepatocytes with or without a PPAR-γ agonist. Wild-type C57BL/6J mice were fed a high-fat (HF) diet and administered rosiglitazone. RESULTS: Primary hepatocytes exhibited significant lipid deposition and increased ROS production after incubation with FFAs. CM from lipid-laden hepatocytes promoted macrophage polarization to the M1 type and activation of the TLR4/NF-κB pathway. A PPAR-γ agonist ameliorated oxidative stress and NLRP3 inflammasome activation in lipid-laden hepatocytes and subsequently prevented M1 macrophage polarization. Hepatocyte-specific PPAR-γ deficiency aggravated oxidative stress and NLRP3 inflammasome activation in lipid-laden hepatocytes, which further promoted M1 macrophage polarization. Rosiglitazone administration improved oxidative stress and NLRP3 inflammasome activation in HF diet-induced NAFLD mice in vivo. CONCLUSION: Upregulation of PPAR-γ activity in hepatocytes alleviated NAFLD by modulating the crosstalk between hepatocytes and macrophages via the reactive oxygen species-NLRP3-IL-1ß pathway.

lncRNA NONRATT013819.2 promotes transforming growth factor-ß1-induced myofibroblastic transition of hepatic stellate cells by miR24-3p/lox.

Long noncoding RNAs (lncRNAs) are key regulators of hepatic stellate cells (HSCs), yet the role of upregulated lncRNA-NONRATT013819.2 in activated HSCs remains uncertain. In this study, the effects of NONRATT013819.2 on proliferation, apoptosis, migration, and contraction of transforming growth factor (TGF)-ß1-induced HSCs were investigated. The mechanisms of NONRATT013819.2 on the activated HSCs were explored by loss-of-function of NONRATT013819.2 and gain-of-function of the target gene. Here, TGF-ß1 treatment resulted in a gradual increase in the expression of cytoskeleton markers (collagen, α-SMA, and TIMP1), NONRATT013819.2, miR24-3p, and lysyl oxidase (Lox) over time in HSCs. NONRATT013819.2 acted as a sponge of miR24-3p to competitively abolish the inhibition of the lox gene in HSCs. Silencing of NONRATT013819.2 suppressed the expression of cytoskeleton markers, proliferation, and the proportion of cells that entered the S-phase, and promoted apoptosis in TGF-ß1-activated HSCs. These effects were reversed when lox overexpression was introduced simultaneously. Similarly, silencing of NONRATT013819.2 also blocked ECM reconstruction, while recused by lox overexpression in TGF-ß1-activated HSCs. In conclusion, upregulation of NONRATT013819.2 promotes the myofibroblastic transition by competitively binding miR24-3p to release lox in HSCs. Therefore, targeted therapy of NONRATT013819.2 may have the potential for liver fibrosis.

Evaluation of controlled attenuation parameter in assessing hepatic steatosis in patients with autoimmune liver diseases.

BACKGROUND: Hepatic steatosis commonly occurs in some chronic liver diseases and may affect disease progression. AIM: To investigate the performance of controlled attenuation parameter (CAP) for the diagnosis of hepatic steatosis in patients with autoimmune liver diseases (AILDs). METHODS: Patients who were suspected of having AILDs and underwent liver biopsy were consistently enrolled. Liver stiffness measurement (LSM) and CAP were performed by transient elastography. The area under the receiver operating characteristic (AUROC) curve was used to evaluate the performance of CAP for diagnosing hepatic steatosis compared with biopsy. RESULTS: Among 190 patients with biopsy-proven hepatic steatosis, 69 were diagnosed with autoimmune hepatitis (AIH), 18 with primary biliary cholangitis (PBC), and 27 with AIH-PBC overlap syndrome. The AUROCs of CAP for the diagnosis of steatosis in AILDS were 0.878 (0.791-0.965) for S1, 0.764 (0.676-0.853) for S2, and 0.821 (0.716-0.926) for S3. The CAP value was significantly related to hepatic steatosis grade (P < 0.001). Among 69 patients with AIH, the median CAP score was 205.63 ± 47.36 dB/m for S0, 258.41 ± 42.83 dB/m for S1, 293.00 ± 37.18 dB/m for S2, and 313.60 ± 27.89 dB/m for S3. Compared with patients with nonalcoholic fatty liver disease (NAFLD) presenting with autoimmune markers, patients with AIH concomitant with NAFLD were much older and had higher serum IgG levels and LSM values. CONCLUSION: CAP can be used as a noninvasive diagnostic method to evaluate hepatic steatosis in patients with AILDs. Determination of LSM combined with CAP may help to identify patients with AIH concomitant with NAFLD from those with NAFLD with autoimmune phenomena.

miR-16 integrates signal pathways in myofibroblasts: determinant of cell fate necessary for fibrosis resolution.

Liver fibrosis is characterized by the transdifferentiation of hepatic stellate cells (HSCs) to myofibroblasts and poor response to treatment. This can be attributed to the myofibroblast-specific resistance to phenotype reversal. In this study, we complemented miR-16 into miR-16-deficient myofibroblasts and analyzed the global role of miR-16 using transcriptome profiling and generating a pathway-based action model underlying transcriptomic regulation. Phenotypic analysis of myofibroblasts and fibrogenic characterization were used to understand the effect of miR-16 on phenotypic remodeling of myofibroblasts. miR-16 expression altered the transcriptome of myofibroblasts to resemble that of HSCs. Simultaneous targeting of Smad2 and Wnt3a, etc. by miR-16 integrated signaling pathways of TGF-ß and Wnt, etc., which underlay the comprehensive regulation of transcriptome. The synergistic effect of miR-16 on the signaling pathways abolished the phenotypic characteristics of myofibroblasts, including collagen production and inhibition of adipogenesis. In vivo, myofibroblast-specific expression of miR-16 not only eliminated mesenchymal cells with myofibroblast characteristics but also restored the phenotype of HSCs in perisinusoidal space. This phenotypic remodeling resolved liver fibrosis induced by chronic wound healing. Therefore, miR-16 may integrate signaling pathways crucial for the fate determination of myofibroblasts. Its global effect induces the reversal of HSC-to-myofibroblast transdifferentiation and, subsequently, the resolution of fibrogenesis. Taken together, these findings highlight the potential of miR-16 as a promising therapeutic target for liver fibrosis.

Performance of transient elastography in assessing liver fibrosis in patients with autoimmune hepatitis-primary biliary cholangitis overlap syndrome.

AIM: To investigate the performance of transient elastography (TE) for diagnosis of fibrosis in patients with autoimmune hepatitis-primary biliary cholangitis (AIH-PBC) overlap syndrome. METHODS: A total of 70 patients with biopsy-proven AIH-PBC overlap syndrome were included. Spearman correlation test was used to analyze the correlation of liver stiffness measurement (LSM) and fibrosis stage. Independent samples Student's t-test or one-way analysis of variance was used to compare quantitative variables. Receiver operating characteristics (ROC) curve was used to calculate the optimal cut-off values of LSM for predicting individual fibrosis stages. A comparison on the diagnostic accuracy for severe fibrosis was made between LSM and other serological scores. RESULTS: Patients with AIH-PBC overlap syndrome had higher median LSM than healthy controls (11.3 ± 6.4 kPa vs 4.3 ± 1.4 kPa, P < 0.01). LSM was significantly correlated with fibrosis stage (r = 0.756, P < 0.01). LSM values increased gradually with an increased fibrosis stage. The areas under the ROC curves of LSM for stages F ≥ 2, F ≥ 3, and F4 were 0.837 (95%CI: 0.729-0.914), 0.910 (0.817-0.965), and 0.966 (0.893-0.995), respectively. The optimal cut-off values of LSM for fibrosis stages F ≥ 2, F ≥ 3, and F4 were 6.55, 10.50, and 14.45 kPa, respectively. LSM was significantly superior to fibrosis-4, glutaglumyl-transferase/platelet ratio, and aspartate aminotransferase-to-platelet ratio index scores in detecting severe fibrosis (F ≥ 3) (0.910 vs 0.715, P < 0.01; 0.910 vs 0.649, P < 0.01; 0.910 vs 0.616, P < 0.01, respectively). CONCLUSION: TE can accurately detect hepatic fibrosis as a non-invasive method in patients with AIH-PBC overlap syndrome.

The immunobiology and clinical features of type 1 autoimmune polyglandular syndrome (APS-1).

Autoimmune Polyglandular Syndrome type 1 (APS-1) is a subtype of the autoimmune polyendocrine syndrome characterized by the simultaneous or sequential dysfunction of multiple endocrine or non-endocrine glands. A clinical diagnosis of APS-1 is typically based on the presence of at least two of three following criteria: chronic mucocutaneous candidiasis, hypoparathyroidism and adrenal insufficiency. The first identified causative mutated gene for APS-1 is autoimmune regulator (AIRE) encoding a critical transcription factor, which is primarily expressed in the medullary thymic epithelial cells (mTECs) for generating central immune tolerance. A wide range of chronic, debilitating complications, with no obvious correlation with genetics, makes a diagnosis of APS-1 challenging early in the disease course. Managing APS-1 is difficult due to its complexity, especially the intricate relationships within manifestations and genetic mutations. The past decades have witnessed dramatic progress in elucidating the function of AIRE and conducting large-scale cohort studies in APS-1. However, no clear evidence-based guidelines have been established in APS-1. In this review, we provide a detailed critical overview of the study history, epidemiology, clinical features, and related mechanisms of autoimmunity in APS-1, as well as currently available therapies for this autoimmune disorder.

RNA Sequencing and Bioinformatics Analysis Implicate the Regulatory Role of a Long Noncoding RNA-mRNA Network in Hepatic Stellate Cell Activation.

BACKGROUND/AIMS: To analyze the long noncoding (lncRNA)-mRNA expression network and potential roles in rat hepatic stellate cells (HSCs) during activation. METHODS: LncRNA expression was analyzed in quiescent and culture-activated HSCs by RNA sequencing, and differentially expressed lncRNAs verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) were subjected to bioinformatics analysis. In vivo analyses of differential lncRNA-mRNA expression were performed on a rat model of liver fibrosis. RESULTS: We identified upregulation of 12 lncRNAs and 155 mRNAs and downregulation of 12 lncRNAs and 374 mRNAs in activated HSCs. Additionally, we identified the differential expression of upregulated lncRNAs (NONRATT012636.2, NONRATT016788.2, and NONRATT021402.2) and downregulated lncRNAs (NONRATT007863.2, NONRATT019720.2, and NONRATT024061.2) in activated HSCs relative to levels observed in quiescent HSCs, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that changes in lncRNAs associated with HSC activation revealed 11 significantly enriched pathways according to their predicted targets. Moreover, based on the predicted co-expression network, the relative dynamic levels of NONRATT013819.2 and lysyl oxidase (Lox) were compared during HSC activation both in vitro and in vivo. Our results confirmed the upregulation of lncRNA NONRATT013819.2 and Lox mRNA associated with the extracellular matrix (ECM)-related signaling pathway in HSCs and fibrotic livers. CONCLUSION: Our results detailing a dysregulated lncRNA-mRNA network might provide new treatment strategies for hepatic fibrosis based on findings indicating potentially critical roles for NONRATT013819.2 and Lox in ECM remodeling during HSC activation.

The role of liver stiffness measurement in the evaluation of liver function and esophageal varices in cirrhotic patients.

OBJECTIVE: We aimed to evaluate the efficacy of liver stiffness measurement (LSM) in predicting the presence and severity of esophageal varices (EV) and investigating its association with liver function (LF) in patients with liver cirrhosis. METHODS: Medical records of 90 cirrhotic patients who underwent LSM by transient elastography were retrospectively reviewed. The relationship between LSM and the presence and severity of EV was evaluated by esophagogastroduodenoscopy (EGD) and multislice spiral computed tomography (MSCT). Another 25 healthy individuals were included as controls. RESULTS: LSM was significantly associated with the Child-Pugh score in cirrhotic patients, with the highest LSM in those with Child-Pugh C. Patients with clinically decompensated cirrhosis had a higher LSM than those with compensated cirrhosis (36.75 ± 16.54 kPa vs 17.65 ± 10.87 kPa, P < 0.01). However, there was no significant difference in LSM value between patients with severe EV and those with no or non-severe EV determined by endoscopy (28.18 ± 17.44 kPa vs 31.00 ± 18.44 kPa) or MSCT (29.71 ± 18.39 kPa vs 24.90 ± 14.80 kPa). The diagnostic value of LSM for predicting severe EV was low in unselected cirrhotic patients. The presence of EV examined by EGD and MSCT was similar to each other. CONCLUSIONS: LSM could be used to evaluate the progression of liver cirrhosis continuously. However, its role in assessing EV grades in advanced cirrhosis needs further confirmation. MSCT can assess EV accurately and may serve as an alternative to endoscopy in the assessment of portal hypertension.

Therapeutic potential of microRNA: a new target to treat intrahepatic portal hypertension?

Intrahepatic portal hypertension accounts for most of the morbidity and mortality encountered in patients with liver cirrhosis, due to increased portal inflow and intrahepatic vascular resistance. Most treatments have focused only on portal inflow or vascular resistance. However, miRNA multitarget regulation therapy may potentially intervene in these two processes for therapeutic benefit in cirrhosis and portal hypertension. This review presents an overview of the most recent knowledge of and future possibilities for the use of miRNA therapy. The benefits of this therapeutic modality--which is poorly applied in the clinical setting--are still uncertain. Increasing the knowledge and current understanding of the roles of miRNAs in the development of intrahepatic portal hypertension and hepatic stellate cells (HSCs) functions, as well as their potential as novel drug targets, is critical.

Dynamic expression of miR-126* and its effects on proliferation and contraction of hepatic stellate cells.

In our previous study, miR-126 was identified as one of the leading miRNAs that is downregulated during activation of hepatic stellate cells (HSCs). However, the roles and related mechanisms of miR-126 in HSCs are not understood. In this study, we compared expression of miR-126 during HSC activation both in vitro and in vivo. We also applied RNA interference to analyze the role and mechanism of miR-126(*) in the activation of HSCs. Restoring HSCs with Lv-miR-126(*) resulted in decreased proliferation, accumulation of extracellular matrix components, and cell contraction, while also negatively regulating the vascular endothelial growth factor (VEGF) signal transduction pathways by partially targeted VEGF-A. Thus, we postulate that miR-126 may be a biological marker for the activation of HSCs, and useful for reducing intrahepatic vascular resistance and improving the sinusoidal microcirculation in chronic liver diseases.

Effects of NF-kappaB inhibitor on titanium particulate-induced inflammation in a murine model.

BACKGROUND: Activation of nuclear factor kappa B (NF-kappaB) signaling in response to implant particulates may be critical in the pathogenesis of implant loosening after joint arthroplasty. The purpose of this study was to investigate the inhibitory effects of pyrrolidine dithiocarbamate (PDTC) in a murine model of inflammation induced by titanium (Ti) particulates. MATERIALS AND METHODS: Ti particulates were introduced into established air pouches on C57BL/6J mice. Mice were injected intraperitoneally with either high-dose PDTC (100 mg/kg) or low-dose PDTC (50 mg/kg). Mice without drug treatment, as well as mice injected with saline alone were included. Each group consisted of sixteen mice. The membranes and lavage fluid were harvested 2 d or 7 d after injection of particulate suspension for histological and molecular analysis. RESULTS: Histologic analysis showed that PDTC reduced inflammatory responses in air pouches, that is, thinner membrane and decreased cellular infiltration. In addition, PDTC reduced the release of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in the lavage fluid or supernatant of homogenates as evaluated by ELISA. CONCLUSION: These results suggest that PDTC inhibits Ti particulate-induced inflammatory responses in the murine model; thus it represents a promising therapeutic candidate for the prevention and treatment of implant loosening.

Effects of upregulated expression of microRNA-16 on biological properties of culture-activated hepatic stellate cells.

In our previous studies, we identified miR-16 as being downregulated during activation of hepatic stellate cells (HSCs) by microarray hybridization. However, the roles and related mechanisms of miR-16 in HSCs are not understood. In this study, The miRNA RNAi technique was used to analyze the effects of miR-16 on biological properties of HSCs in vitro. The lentiviral vector encoding miR-16 was constructed and transfected. Furthermore, the expression level of miR-16 was measured by real-time PCR. Cellular growth and proliferation capacity were assayed using the cell counting kit-8 (CCK-8). The apoptosis rate and cell-cycle distribution were measured by flow cytometry. Cell morphological characteristics were identified by phase-contrast microscopy, fluorescence microscopy and electron microscopy. The underlying mechanisms related to the changes in biological properties were assessed. The identity of the recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. Virus titer was 10(8) > ifu/m. Restoring the intracellular miRNAs by miR-16 administration greatly reduced the expression levels of cyclin D1 (CD1). Cell-cycle arrest and typical features of apoptosis were detected in activated HSCs treated with pLV-miR-16. Our results indicate that transduction of miR-16 offers a feasible approach to significantly inhibit HSC proliferation and increase the apoptosis index. Thus, targeted transfer of miR-16 into HSC may be useful for the treatment of hepatic fibrosis.

Changes in microRNAs associated with hepatic stellate cell activation status identify signaling pathways.

Activation of hepatic stellate cells (HSCs), which is regulated by multiple signal transduction pathways, is the key event in liver fibrosis. Moreover, members of these pathways are important targets for microRNAs (miRNAs). To better understand the critical pathways of HSC activation, we performed comprehensive comparative bioinformatics analysis of microarrays of quiescent and activated HSCs. Changes in miRNAs associated with HSC activation status revealed that 13 pathways were upregulated and 22 pathways were downregulated by miRNA. Furthermore, mitochondrial integrity, based on highly upregulated Bcl-2 and downregulated caspase-9, was confirmed in HSCs and fibrotic livers by immnofluorescence assay, quantitative RT-PCR, and western blot analysis. These findings provide in vitro and in vivo evidence that the mitochondrial pathway of apoptosis plays a significant role in the progression of liver fibrogenesis via HSC activation.

miR-15b and miR-16 are implicated in activation of the rat hepatic stellate cell: An essential role for apoptosis.

BACKGROUND/AIMS: To reveal the microRNA (miRNA) expression profile and related roles in rat HSCs during activation. METHODS: miRNA expression profiling was analyzed in quiescent and in culture-activated HSCs by microarray. The differentially expressed miRNAs, as verified by RT-PCR, were subjected to gene ontology (GO) analysis. Furthermore, the effects of miR-16 and miR-15b on the apoptosis of activated HSCs were investigated by Hoechst 33258, TUNEL staining and annexin-V/PI labeling flow cytometry. The underlying mechanism related to Bcl-2 and caspases was assessed. RESULTS: The upregulated and downregulated miRNAs in activated HSCs were 12 miRNAs and 9 miRNAs, respectively. The differential expression of miR-16, -15b, -122, -138, -143, and -140 was validated. High-enrichment GOs containing apoptosis-related targeted genes and miRNA-gene networks characterized by Bcl-2, which was targeted by the miR-15/16 family, uncovered the critical role of miR-16 and miR-15b in apoptosis. Restoring the intracellular miRNAs by miR-16 and miR-15b administration greatly reduced Bcl-2, and increased the expression of caspases 3, 8, and 9. Significantly elevated rates of apoptosis were then induced in activated HSCs. CONCLUSIONS: The activation of HSCs relate to 21 miRNAs. Among these, mir-15b and miR-16 may be essential for apoptosis by targeting Bcl-2 and the caspase signaling pathway.

Effect of qi-tonifying and stasis-eliminating therapy on expression of vascular endothelial growth factor and its receptors Flt-1, Flk-1 in the brain of intracerebral hemorrhagic rats.

OBJECTIVE: To investigate the effects and mechanism of qi-tonifying and stasis-eliminating (QTSE) therapy on the expression of vascular endothelial growth factor (VEGF) and its receptors Flt-1 and Flk-1 in the brains of intracerebral hemorrhagic (model) rats. METHODS: One hundred and eighty Sprague-Dawley rats were randomly divided into six groups: the normal group (n=5), the sham-operative (SO) group (n=35), the model group (n=35), the QTSE group (n=35), the QT group (n=35) and the SE group (n=35). All the rats except those in the normal group and SO group were established into an intracerebral hemorrhage(ICH) model by intracerebral injection of collagenase type VII and the latter three were orally administered with Buyang Huanwu Decoction (a classical recipe for QTSE) or with some of its components for qi-tonification and for stasis-elimination, respectively. To the other three groups, normal saline solutions were given instead. Behavioral tests were carried out in the animals randomly chosen from each group on days 1, 2, 4, 7, 14, 21 and 28 after modeling. The expressions of VEGF, Flk-1 and Flt-1 were determined by immunohistochemistry and the number of vascular segments with positive expression in the injured brain area of the rats was calculated. RESULTS: From day 7 onwards, the asymmetric forelimb use rate in the QTSE group recovered more significantly than that in the other model groups. In the model group, the expressions of VEGF, Flk-1 and Flt-1 appeared on day 1 and reached a peak on day 21, then weakened gradually. In the QTSE group, as compared with the other model groups, a higher level of VEGF expression was shown from day 7 (P<0.01) and a higher level of Flt-1 expression was shown from the 7th day to the 21st day (P<0.01). CONCLUSION: QTSE therapy can up-regulate the expressions of VEGF and its receptors (Flk-1 and Flt-1) and improve the recovery of kinetic function in the ICH rats, which may be correlated with its action in modulating vascular regeneration to promote the reconstruction of microvascular networks in the damaged areas.