Pig H3K4me3, H3K27ac, and gene expression profiles reveal reproductive tissue-specific activity of transposable elements.

Regulatory sequences and transposable elements (TEs) account for a large proportion of the genomic sequences of species; however, their roles in gene transcription, especially tissue-specific expression, remain largely unknown. Pigs serve as an excellent animal model for studying genomic sequence biology due to the extensive diversity among their wild and domesticated populations. Here, we conducted an integrated analysis using H3K27ac ChIP-seq, H3K4me3 ChIP-seq, and RNA-seq data from 10 different tissues of seven fetuses and eight closely related adult pigs. We aimed to annotate the regulatory elements and TEs to elucidate their associations with histone modifications and mRNA expression across different tissues and developmental stages. Based on correlation analysis between mRNA expression and H3K27ac and H3K4me3 peak activity, results indicated that H3K27ac exhibited stronger associations with gene expression than H3K4me3. Furthermore, 1.45% of TEs overlapped with either the H3K27ac or H3K4me3 peaks, with the majority displaying tissue-specific activity. Notably, a TE subfamily (LTR4C_SS), containing binding motifs for SIX1 and SIX4, showed specific enrichment in the H3K27ac peaks of the adult and fetal ovaries. RNA-seq analysis also revealed widespread expression of TEs in the exons or promoters of genes, including 4 688 TE-containing transcripts with distinct development stage-specific and tissue-specific expression. Of note, 1 967 TE-containing transcripts were enriched in the testes. We identified a long terminal repeat (LTR), MLT1F1, acting as a testis-specific alternative promoter in SRPK2 (a cell cycle-related protein kinase) in our pig dataset. This element was also conserved in humans and mice, suggesting either an ancient integration of TEs in genes specifically expressed in the testes or parallel evolutionary patterns. Collectively, our findings demonstrate that TEs are deeply embedded in the genome and exhibit important tissue-specific biological functions, particularly in the reproductive organs.

Improving ocular bioavailability of hydrophilic drugs through dynamic covalent complexation.

The clinical benefits of diquafosol tetrasodium (DQS), a hydrophilic P2Y2 receptor agonist for dry eye, have been hindered by a demanding dosing regimen. Nevertheless, it is challenging to achieve sustained release of DQS with conventional drug delivery vehicles which are mainly designed for hydrophobic small molecule drugs. To address this, we developed an affinity hydrogel for DQS by taking advantage of borate-mediated dynamic covalent complexation between DQS and hydroxypropyl guar. The resultant formulation (3% DQS Gel) was characterized by sustained release, low corneal permeation, and extended ocular retention, which were desirable attributes for ocular surface drug delivery. Both in vitro and in vivo studies had been carried out to verify the biocompatibility of 3% DQS Gel. Using corneal fluorescein staining, the Schirmer's test, PAS staining, quantitative PCR and immunohistological analyses as outcome measures, the superior therapeutic effects of 3% DQS Gel over PBS, the hydrogel vehicle and free DQS were demonstrated in a mouse dry eye model. Our DQS delivery strategy reported herein is readily applicable to other hydrophilic small molecule drugs with cis-diol moieties, thus providing a general solution to improve clinical outcomes of numerous diseases.

A20 ameliorates disc degeneration by suppressing mTOR/BNIP3 axis-mediated mitophagy.

BACKGROUND: The pathological mechanism of intervertebral disc degeneration (IDD) is an unanswered question that we are committed to exploring. A20 is an anti-inflammatory protein of nucleus pulposus (NP) cells and plays a protective role in intervertebral disc degeneration. OBJECTIVE: This study aims to investigate the molecular mechanism by which A20 attenuates disc degeneration. METHODS: The proteins of interest were measured by immunoblotting, immunofluorescence, ELISA assay, and immunohistochemical technique to conduct related experiments. Immunofluorescence assays and mitochondrial membrane potential (JC-1) were used to assess mitophagy and mitochondrial fitness, respectively. RESULTS: Here, we demonstrated that A20 promoted mitophagy, attenuated pyroptosis, and inhibited the degradation of the extracellular matrix, consequently significantly ameliorating disc degeneration. Mechanistically, A20 reduces pyroptosis and further suppresses cellular mTOR activity. On the one hand, A20-induced mTOR inhibition triggers BNIP3-mediated mitophagy to ensure mitochondrial fitness under LPS stimulation, as a result of mitigating mitochondrial dysfunction induced by LPS. On the other hand, A20-induced mTOR inhibition reduces the loss of mitochondrial membrane potential and the generation of Mitochondrial ROS. CONCLUSION: The study revealed that A20 promotes BNIP3-mediated mitophagy by suppressing mTOR pathway activation against LPS-induced pyroptosis.

CircRNA-Associated ceRNA Network Reveals Focal Adhesion and Metabolism Pathways in Neuropathic Pain.

Background: Increasing evidence has shown that noncoding RNAs perform a remarkable function in neuropathic pain (NP); nonetheless, the mechanisms underlying the modulation of competitive endogenous RNA in NP remain uncertain. The goal of this research was to investigate the molecular processes underlying NP. Methods: We utilized the Gene Expression Omnibus (GEO) to obtain NP-related microarray datasets that included the expression patterns of circular RNAs (circRNAs) and messenger RNAs (mRNAs). Following that, bioinformatics analyses and a molecular biology experiment were carried out. Results: According to the findings, carrying out enrichment studies of the targeted genes had an impact on a variety of NP-related pathways. Notably, we isolated a ceRNA subnetwork incorporating two upregulated circRNAs (Esrrg and Map3k3) which primarily participate in the focal adhesion pathway by regulating Integrin Subunit Beta 4 (ITGB4) and two downregulated circRNAs (Dgkb and Atp2a2), which potentially regulate metabolism-related molecule Lipase A (LIPA). Conclusions: According to our findings, the focal adhesion and metabolic signaling pathways could be critical in the advancement of NP, and some circRNA might regulate this biological process through the ceRNA network, which might offer pertinent insights into the underlying mechanisms.

A20 attenuates pyroptosis and apoptosis in nucleus pulposus cells via promoting mitophagy and stabilizing mitochondrial dynamics.

BACKGROUND: A20 is an anti-inflammatory molecule in nucleus pulposus (NP) cells. The anti-inflammatory properties of A20 are mainly attributed to its ability to suppress the NF-κB pathway. However, A20 can protect cells from death independently of NF-κB regulation. This study aimed to investigate the effects of A20 on pyroptosis and apoptosis of NP cells induced by lipopolysaccharide (LPS). METHODS: NP cells induced by LPS were used as an in vitro model of the inflammatory environment of the intervertebral disc. Pyroptosis, apoptosis, and mitophagy marker proteins were detected. Then, NP cells were transfected with A20 overexpressed lentivirus or A20-siRNA. Annexin V FITC/PI, Western blotting, and immunofluorescence assays were used to detect the apoptosis, pyroptosis, and mitophagy of NP cells. Furthermore, the expressions of A20, related proteins, and related inflammatory cytokines were detected by western blotting, and ELISA. RESULTS: Apoptosis and pyroptosis of NP cells increased gradually treated with LPS for 12 h, 24 h, and 48 h. Differently, the level of mitophagy increased first and then decreased, and was the highest at LPS treatment for 12 h. Overexpression or knockdown of A20 in NP cells revealed that A20 attenuated the pyroptosis, apoptosis, and production of inflammatory cytokines of NP cells induced by LPS, while A20 sponsored mitophagy, reduced ROS production and collapse of mitochondrial membrane potential (ΔΨm). Moreover, A20 also promoted mitochondrial dynamic homeostasis and attenuated LPS-induced excessive mitochondrial fission. Excitingly, inhibition of mitophagy attenuated the effect of A20 on the negative regulation of pyroptosis of NP cells induced by LPS. Pyroptosis was accompanied by a large release of inflammatory cytokines. Inhibition of pyroptosis also significantly reduced apoptosis of NP cells. Finally, The mitochondria-targeted active peptide SS-31 inhibited LPS-induced pyroptosis and ROS production in NP cells. CONCLUSIONS: To sum up, A20 attenuates pyroptosis and apoptosis of NP cells via promoting mitophagy and stabilizing mitochondrial dynamics. Besides, A20 reduces LPS-induced NP cell apoptosis by inhibiting NLRP3 inflammasome-mediated pyroptosis. It provides theoretical support for the reduction of functional NP cell loss in the intervertebral disc through the gene-targeted intervention of A20.

Underlying Mechanisms and Related Diseases Behind the Complex Regulatory Role of NOD-Like Receptor X1.

Among nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), NOD-like receptor X1 (NLRX1) is the only known NLR family member that is targeted to the mitochondria, which contains a C-terminal leucine-rich repeat domain, a central conserved nucleotide-binding domain, and an unconventional N-terminal effector domain. It is unique due to several atypical features, such as mitochondrial localization, noninflammasome forming, and relatively undefined N-terminal domain. NLRX1 has multiple functions, including negative regulation of type-I interferon signaling, attenuation of proinflammatory nuclear factor kappa B (NF-κB) signaling, autophagy induction, modulation of reactive oxygen species production, cell death regulation, and participating in cellular senescence. In addition, due to its diverse functions, NLRX1 has been associated with various human diseases, including respiratory, circulatory, motor, urinary, nervous, and digestive systems, to name but a few. However, the exact regulatory mechanisms of NLRX1 are still unclear in many related diseases since conflicting and controversial topics on NLRX1 in the previous studies remain. In this review, we review recent research advances on the underlying mechanisms and related disorders behind the complex regulatory role of NLRX1, which may provide a promising target to prevent and/or treat the corresponding diseases.

Small extracellular vesicles from hypoxic mesenchymal stem cells alleviate intervertebral disc degeneration by delivering miR-17-5p.

Minimally invasive repair strategies are a very promising approach for the treatment of intervertebral disc degeneration (IDD). In recent years, small extracellular vesicles (sEVs) secreted from mesenchymal stem cells (MSCs) have been shown great potential in alleviating IDD. However, in vitro experiments, MSCs are usually exposed to a normoxic micro-environment, which differs greatly from the hypoxic micro-environment in vivo. The primary purpose of our research was to determine whether sEVs isolated from MSCs under hypoxic status (H-sEVs) exhibit a more beneficial effect on protecting IDD compared with sEVs derived from MSCs under normoxic status (N-sEVs). A tail IDD rat model and a series of experiments in vitro were conducted to compare the beneficial effects of PBS, N-sEVs, and H-sEVs treatment. Then, to validate the role of sEVs miRNAs in IDD, a miRNA microarray sequencing analysis and a series of rescue experiments were conducted. Luciferase activity, RNA-ChIP and western blot were performed to explore the potential mechanisms. The results indicate that sEVs alleviate IDD by ameliorating the homeostatic imbalance between anabolism and catabolism in vivo and in vitro. Microarray sequencing result shows that miR-17-5p is maximally enriched in H-sEVs. Toll-like receptor 4 (TLR4) was determined to be a target downstream gene of miR-17-5p. Finally, it was found that H-sEVs miR-17-5p may modulate proliferation and synthesis of human nucleus pulposus cells (HNPCs) matrix via TLR4 pathway. In conclusion, H-sEVs miR-17-5p alleviate IDD via promoting HNPCs matrix proliferation and synthesis, providing new therapeutic targets for IDD. STATEMENT OF SIGNIFICANCE: Intervertebral disc degeneration (IDD) is the primary cause of low back pain (LBP), which is a huge burden to society. Our research demonstrates for the first time that hypoxic pretreatment of small extracellular vesicles (H-sEVs) effectively alleviated the progress of IDD. In short, in the present research, we found that H-sEVs miR-17-5p could modulate proliferation and synthesis of nucleus pulposus cells (NPCs) matrix via TLR4/PI3K/AKT pathway. Therefore, hypoxic pre-treatment is a prospective and efficient method to optimize the therapeutic effect of MSCs-derived sEVs. miRNA and MSCs-derived sEVs combination may be a promising therapeutic approach for IDD.

The mitochondrial antioxidant SS-31 attenuated lipopolysaccharide-induced apoptosis and pyroptosis of nucleus pulposus cells via scavenging mitochondrial ROS and maintaining the stability of mitochondrial dynamics.

Evidence has shown that effects from inflammation and mitochondrial dysfunction lead to pyroptosis and apoptosis of nucleus pulposus (NP) cells. Damaged mitochondria release dangerous molecules such as reactive oxygen species (ROS), activating the NLRP3 inflammasome. SS-31 is a mitochondria-targeting peptide that has been used in the treatment of many diseases by scavenging ROS and ameliorating mitochondrial function. This study found that SS-31 ameliorated lipopolysaccharide (LPS)-induced loss of cell viability, ROS production, and apoptosis in NP cells. Moreover, mitochondrial dynamics and ATP synthesis were restored on pretreatment with SS-31 compared with the LPS group. For the molecular mechanism research, SS-31 stabilized mitochondrial morphology and inhibited the activation of the NF-κB pathway and the activation of the NLRP3 inflammasome. To evaluate whether the inhibition of NLRP3 inflammasome activation by SS-31 is dependent on the clearance of mitochondrial ROS, we comparatively analyzed the activation of NLRP3 inflammasome in NP cells pretreated with SS-31 and the ROS scavenger N-acetyl-L-cysteine (NAC). The results indicate that SS-31 could inhibit NLRP3 inflammasome activation by limiting the production of mitochondrial ROS. To sum up, our results revealed that SS-31 inhibits LPS-induced apoptosis, pyroptosis, and inflammation in NP cells via scavenging ROS and maintaining the stability of mitochondrial dynamics, which could be considered a promising therapeutic intervention for disk degeneration.

Risk Factors for Increased Surgical Drain Output After Transforaminal Lumbar Interbody Fusion.

OBJECTIVE: To investigate the risk factors for increased surgical drain output after transforaminal lumbar interbody fusion (TLIF). METHODS: Patients who underwent TLIF in a single center from June 2017 to January 2020 were included in this study. They were divided into the increased surgical drain output group and no increased surgical drain output group according to the boundary of the median drain output. Patients' demographic and clinical parameters were compared between the 2 groups. Risk factors for increased surgical drain output were identified by univariate and multivariate logistic regression analysis. RESULTS: This study enrolled 368 patients who underwent TLIF. Among them, 187 patients had increased surgical drain output (drain output ≥50th percentile or 480 mL). Univariate analysis showed that age (P < 0.001), smoking status (P = 0.002), number of fused levels (P < 0.001), intraoperative blood loss (P < 0.001), intraoperative end plate injury (P < 0.001), administration of tranexamic acid (TXA) (P = 0.002), and surgical duration (P < 0.001) were significantly associated with increased surgical drain output. Multiple logistic regression analysis revealed that older age (P = 0.001), smoking (P = 0.005), more fused levels (P < 0.001), and intraoperative end plate injury (P = 0.017) were the independent risk factors, while administration of TXA (P = 0.012) was a protective factor. CONCLUSIONS: This study showed that older age, smoking, more fused levels, and intraoperative end plate injury were the independent risk factors, while administration of TXA was a protective factor for increased surgical drain output after TLIF.

Influence of Characteristics of Thermoplastic Polyurethane on Graphene-Thermoplastic Polyurethane Composite Film.

Graphene-thermoplastic polyurethane (G-TPU) composite films were fabricated by traditional blending method and tape casting process with commercial graphene sheets as functional fillers and TPU masterbatches of four different melting points as matrix, respectively. The effects of matrix on the distribution of graphene, the electrical conductivity, and infrared (IR) light thermal properties of the G-TPU composite films were investigated. The experimental results reveal that the characteristics of TPU has little influence on the electrical conductivity of the G-TPU composite films, although the four TPU solutions have different viscosities. However, under the same graphene mass content, the thermal conductivity of four G-TPU composite films with different melting points is significantly different. The four kinds of G-TPU composite films have obvious infrared (IR) thermal effect. There is little difference in the temperatures between the composite films prepared by TPU with melting a point of 100 °C, 120 °C, and 140 °C, respectively; however, when the content of graphene is less than 5 wt%, the temperature of the composite film prepared by TPU with a melting point of 163 °C is obviously lower than that of the other three composite films. The possible reason for this phenomenon is related to the structure of TPU.

A thermophilic chitinase 1602 from the marine bacterium Microbulbifer sp. BN3 and its high-level expression in Pichia pastoris.

Chitinases play an important role in many industrial processes, including the preparation of oligosaccharides with potential applications. In the present study, a 1,713 bp gene of Chi1602, derived from a marine bacterium Microbulbifer sp. BN3, encoding a GH18 family chitinase, was expressed at high levels in Pichia pastoris. Distinct from most of the marine chitinases, the recombinant chitinase 1602 exhibited maximal activity at 60 °C and over a broad pH range between 5.0 and 9.0, and was stable at 50 °C and over the pH range 4.0-9.0. The hydrolytic products derived from colloidal chitins comprised mainly (GlcNAc)2 and GlcNAc, indicating that rChi1602 is a GH18 processive chitinase in conformity with its hypothetical structure. However, rChi1602 showed traces of ß-N-acetylglucosaminidase activity on substrates such as powder chitin, chitosan, and ethylene glycol chitin. The thermophilic rChi1602, which manifests adaptation to a wide pH range and can be expressed at a high level in P. pastoris, is advantageous for applications in industrial processes.

GIT1 contributes to autophagy in osteoclast through disruption of the binding of Beclin1 and Bcl2 under starvation condition.

Approximately 10-15% of all bone fractures do not heal properly, causing patient morbidity and additional medical care expenses. Therefore, better mechanism-based fracture repair approaches are needed. In this study, a reduced number of osteoclasts (OCs) and autophagosomes/autolysosomes in OC can be observed in GPCR kinase 2-interacting protein 1 (GIT1) knockout (KO) mice on days 21 and 28 post-fracture, compared with GIT1 wild-type (GIT1 WT) mice. Furthermore, in vitro experiments revealed that GIT1 contributes to OC autophagy under starvation conditions. Mechanistically, GIT1 interacted with Beclin1 and promoted Beclin1 phosphorylation at Thr119, which induced the disruption of Beclin1 and Bcl2 binding under starvation conditions, thereby, positively regulating autophagy. Taken together, the findings suggest a previously unappreciated role of GIT1 in autophagy of OCs during fracture repair. Targeting GIT1 may be a potential therapeutic approach for bone fractures.

GPCR kinase 2-interacting protein-1 protects against ischemia-reperfusion injury of the spinal cord by modulating ASK1/JNK/p38 signaling.

GPCR kinase 2-interacting protein-1 (GIT1) is a scaffold protein that plays an important role in cell adaptation, proliferation, migration, and differentiation; however, the role of GIT1 in the regulation of neuronal death after spinal cord injury remains obscure. Here, we demonstrate that GIT1 deficiency remarkably increased neuronal apoptosis and enhanced JNK/p38 signaling, which resulted in stronger motor deficits by ischemia-reperfusion in vivo, consistent with the finding of oxygen-glucose deprivation/reoxygenation-induced neuronal injury in vitro. After treatment with JNK and p38 inhibitors, abnormally necroptotic cell death caused by GIT1 knockdown could be partially rescued, with the recovery of neuronal viability, which was still poorer than that in control neurons. Meanwhile, overactivation of JNK/p38 after GIT1 depletion was concomitant with excessive activity of apoptosis signal-regulating kinase-1 (ASK1) that could be abolished by ASK1 silencing in HEK293T cells. Finally, GIT1 could disrupt the oligomerization of ASK1 via interaction between the synaptic localization domain that contains the coiled-coil (CC)-2 domain of GIT1 and the C-terminal CC domain of ASK1. It suppressed the autophosphorylation of ASK1 and led to decreasing activity of the ASK1/JNK/p38 pathway. These data reveal a protective role for GIT1 in neuronal damage by modulating ASK1/JNK/p38 signaling.-Chen, J., Wang, Q., Zhou, W., Zhou, Z., Tang, P.-Y., Xu, T., Liu, W., Li, L.-W., Cheng, L., Zhou, Z.-M., Fan, J., Yin, G.-Y. GPCR kinase 2-interacting protein-1 protects against ischemia-reperfusion injury of the spinal cord by modulating ASK1/JNK/p38 signaling.

How do different types of community commitment influence brand commitment? The mediation of brand attachment.

Although previous research indicates that participation in a brand community may foster consumer loyalty to the brand in question, research has seldom examined the mediating effect of community commitment on brand commitment. Drawing from the typologies of organizational commitment, we divide community commitment into three components: continuance community commitment (continuance CC), affective community commitment (affective CC), and normative community commitment (normative CC). We then assess the mediating role of brand attachment in the relationship between these three components and brand commitment. We test the hypotheses using a sample of online mobile phone brand communities in China. The empirical results reveal that brand attachment exerts an indirect (but not mediated) effect on the relationship between continuance CC and brand commitment and on the relationship between normative CC and brand commitment. We also find that it exerts a partial mediating effect on the relationship between affective CC and brand commitment. The findings contribute to the branding literature and have important implications for brand community management.

Stability and in vivo evaluation of pullulan acetate as a drug nanocarrier.

To develop pullulan acetate nanoparticles (PANs) as a drug nanocarrier, pullulan acetate (PA) was synthesized and characterized. Its acetylation degree determined by the proton nuclear magnetic resonance ((1)H NMR) was 2.6. PANs were prepared by the solvent diffusion method and characterized by transmission electron microscope (TEM), size distribution, and zeta potential techniques. PANs had nearly spherical shape with a size range of 200-450 nm and low zeta potentials both in distilled water and in 10% FBS. The storage stability of PANs was observed in distilled water. PANs were stored for at least 2 months with no significant size and zeta potential changes. The safety of PANs was studied through single dose toxicity test in mice, and the result showed that PANs were well tolerated at the dose of 200 mg/kg in mice. Epirubicin-loaded PANs (PA/EPI) were also prepared and characterized in this study. Moreover, the in vivo pharmacokinetics of PA/EPI was investigated. Compared with the free EPI group, the PA/EPI group exhibited higher plasma drug concentration, longer half-life time (t(1/2)) and the larger area under the curve (AUC). All results suggested that PANs were stable, safe, and showed a promising potential on improving the bioavailability of the loaded drug of the encapsulated drug.

Preparation of folate-modified pullulan acetate nanoparticles for tumor-targeted drug delivery.

The purpose of this work was to develop a novel nano-carrier with targeting property to tumor. In this study, pullulan acetate (PA) was synthesized by the acetylation of pullulan to simplify the preparation technique of nanoparticles. Folic acid (FA) was conjugated to PA in order to improve the cancer-targeting activity. The products were characterized by proton nuclear magnetic resonance (¹H NMR) spectroscopy. Epirubicin-loaded nanoparticles were prepared by a solvent diffusion method. The loading efficiencies and EPI content increased with the amount of triethylamine (TEA) increasing in some degree. FPA nanoparticles could incorporate more epirubicin than PA nanoparticles. The folate-modified PA nanoparticles (FPA/EPI NPs) exhibited faster drug release than PA nanoparticles (PA/EPI NPs) in vitro. Confocal image analysis and flow cytometry test revealed that FPA/EPI NPs exhibited a greater extent of cellular uptake than PA/EPI NPs against KB cells over-expressing folate receptors on the surface. FPA/EPI NPs also showed higher cytotoxicity than PA/EPI NPs. The cytotoxic effect of FPA/EPI NPs to KB cells was inhibited by an excess amount of folic acid, suggesting that the binding and/or uptake were mediated by the folate receptor.

Pullulan acetate nanoparticles prepared by solvent diffusion method for epirubicin chemotherapy.

Pullulan acetate (PA) was synthesized by the reaction of pullulan with acetic anhydride in the presence of pyridine. PA was characterized by Fourier transform infrared (FT-IR) and proton nuclear magnetic resonance ((1)H NMR). A solvent diffusion method was employed in the current work to prepare PA nanoparticles. This technique had some advantages compared with other methods. The particle size increased from 185.7 nm to 423.0 nm with the degree of acetylation increasing from 2.71 to 3.0. Drug-loaded PA nanoparticles were prepared for controlled release of epirubicin (EPI). The drug entrapment and drug content increased with the degree substitution of PA increasing. EPI was released from the nanoparticles in a biphasic profile with a fast release rate in the first 10h followed by a slow release in vitro. A higher cytotoxicity against KB cells was found for EPI-loaded PA nanoparticles in comparison with free EPI. Confocal laser scanning microscopy (CLSM) observations indicate that EPI-loaded nanoparticles were internalized and released in the cytoplasmic compartment.

[Treatment of hepatocellular carcinoma in mice with locally administered epirubicin-loaded poly(D, L)-lactic acid microspheres].

OBJECTIVE: To study the effectiveness of treating hepatocellular carcinoma (HCC) in mice with locally administered epirubicin-loaded poly( D, L) - lactic acid microspheres (EPI-PLA-MS ). METHODS: EPI-PLA-MS was prepared with double emulsion solvent evaporation technique. Five groups of mice (n = 8 in each group) were intraperitoneally injected with five different doses of free epirubicin (FEPI), and the maximum tolerated dose (MTD) was calculated. Then 15 mice with transplanted subcutaneous H22 HCC were divided into three groups (n = 5), which were respectively intratumorally injected with normal saline (NS), blank microspheres, and EPI-PLA-MS (with 9 mg/kg of EPI). After two weeks the tumors were excised and weighed. Another 15 mice with transplanted H22 ascites HCC were divided into three groups (n = 5), which were intraperitonealy injected with the same drugs, and the increased life span were registered exactly. RESULTS: The MTD of intraperitoneally injected FEPI was 9 mg/kg. The tumour-inhibiting rates was 40.35% and 36.09% when EPI-PLA-MS were administered by intratumoral injection to the mice with subcutaneous H22 HCC. It significantly prolonged the survival time of mice with H22 ascites HCC and the increased life span by 153.49% and 142.22% when EPI-PLA-MS were intraperitoneally administered. CONCLUSION: EPI-PLA-MS is a new sustained-release preparation with high-efficacy and low-toxicity in treating HCC and has shown promising prospects when administered locally.