Anti-apoptotic effects of diosgenin on ovariectomized hearts.

BACKGROUND: The anti-apoptotic effects of diosgenin, a steroid saponin, on hearts in female with estrogen deficiency have been less studied. This study aimed to evaluate the anti-apoptotic effects of diosgenin on cardiac widely dispersed apoptosis in a bilateral ovariectomized animal model. METHODS: A total of 60 female Wistar rats, aged 6-7 months, were divided into the sham-operated group (Sham), bilateral ovariectomized rats for 2 months, and ovariectomized rats administered with 0, 10, 50, or 100 mg/kg diosgenin daily (OVX, OVX 10, OVX 50, and OVX 100, respectively) in the second month. The excised hearts were analyzed by H&E staining, TUNEL(+) assays and Western Blot. RESULT: Cardiac TUNEL(+) apoptotic cells, the levels of Fas ligand, Fas death receptors, Fas-associated death domain, active caspase-8, and active caspase-3 (FasL/Fas-mediated pathways) as well as the levels of Bax, Bad, Bax/Bcl2, Bad/p-Bad, cytosolic Cytochrome c, active caspase-9, and active caspase-3 (mitochondria-initiated pathway) were increased in OVX compared with Sham group but those were decreased in OVX 50 compared with OVX. CONCLUSION: Diosgenin appeared to prevent or suppress ovariectomy-induced cardiac FasL/Fas-mediated and mitochondria-initiated apoptosis. These findings might provide one of the possible therapeutic approaches of diosgenin for potentially preventing cardiac apoptosis in women after bilateral ovariectomy or women with estrogen deficiency.

Angiotensin II receptor blocker irbesartan attenuates sleep apnea-induced cardiac apoptosis and enhances cardiac survival and Sirtuin 1 upregulation.

BACKGROUND: The purpose of this study was to investigate whether or not angiotensin II type 1 receptor blocker irbesartan (ARB) with a partial agonist of PPAR-γ could protect against chronic nocturnal intermittent hypoxia (CIH)-induced cardiac Fas/FasL-mediated to mitochondria-mediated apoptosis. METHODS: Sprague-Dawley rats were in a normoxic control group (CON-G), or rats were in a chronic nocturnal intermittent hypoxia group (HP-G, from 3 to 7% oxygen versus 21% oxygen per forty seconds cycle, nocturnally 8 h per day for 1 month), or rats were in a chronic nocturnal intermittent hypoxia group pretreated with ARB (50 mg/kg/day, S.C.) (ARB-HP-G). Echocardiography, H&E staining, TUNEL staining, and Western blotting were measured in the left ventricle. RESULTS: Hypoxia-induced SIRT1 degradation, Fas receptors, FADD, active caspase-8 and caspase-3 (Fas/FasL apoptotic pathway) and Bax, tBid, active caspase-9 and -3 (mitochondrial apoptotic pathway) and TUNEL-positive apoptosis were reduced in ARB-HP-G when compared with HP-G. IGF-I, IGF1 receptor, p-PI3k, p-Akt, Bcl2, and Bcl-XL (IGF1/PI3K/AKT pro-survival pathway) were increased in ARB-HP-G compared to HP-G. CONCLUSIONS: Our findings suggest that the ARB may prevent cardiac Fas/FasL to mitochondrial apoptotic pathways and enhance cardiac IGF1/PI3K/AKT pro-survival pathway in the sleep apnea model associated with JNK de-activation and SIRT1 upregulation. ARB prevents chronic sleep apnea-enhanced cardiac apoptosis via enhancing survival pathways.

Cerebral Cortex Apoptosis in Early Aged Hypertension: Effects of Epigallocatechin-3-Gallate.

This study aimed to investigate cerebral cortex apoptosis on the early aged hypertension and the effects of green tea flavonoid epigallocatechin-3-gallate (EGCG). Twenty-four rats were divided into three groups: a control Wistar-Kyoto group (WKY, n = 8), a spontaneously early aged hypertensive group (SHR, n = 8), and an early aged hypertension with EGCG treatment group (SHR-EGCG, n = 8; daily oral EGCG 200 mg/kg-94%, 12 weeks). At 48 weeks old, blood pressures (BPs) were evaluated and cerebral cortexes were isolated for TUNEL assay and Western blotting. Systolic, diastolic, and mean blood pressure levels in the SHR-EGCG were reduced compared to the SHR. The percentage of neural cell deaths, the levels of cytosolic Endonuclease G, cytosolic AIF (Caspase-independent apoptotic pathway), Fas, Fas Ligand, FADD, Caspase-8 (Fas-mediated apoptotic pathway), t-Bid, Bax/Bcl-2, Bak/Bcl-xL, cytosolic Cytochrome C, Apaf-1, Caspase-9 (Mitochondrial-mediated apoptotic pathway), and Caspase-3 (Fas-mediated and Mitochondria-mediated apoptotic pathways) were increased in the SHR relative to WKY and reduced in SHR-EGCG relative to SHR. In contrast, the levels of Bcl-2, Bcl-xL, p-Bad, 14-3-3, Bcl-2/Bax, Bcl-xL/Bak, and p-Bad/Bad (Bcl-2 family-related pro-survival pathway), as well as Sirt1, p-PI3K/PI3K and p-AKT/AKT (Sirt1/PI3K/AKT-related pro-survival pathway), were reduced in SHR relative WKY and enhanced in SHR-EGCG relative to SHR. In conclusion, green tea flavonoid epigallocatechin-3-gallate (EGCG) might prevent neural apoptotic pathways and activate neural survival pathways, providing therapeutic effects on early aged hypertension-induced neural apoptosis.

LncRNA MYLK-AS1 facilitates tumor progression and angiogenesis by targeting miR-424-5p/E2F7 axis and activating VEGFR-2 signaling pathway in hepatocellular carcinoma.

BACKGROUND: Long non-coding RNAs (lncRNAs) are crucial in the invasion, angiogenesis, progression, and metastasis of hepatocellular carcinoma (HCC). The lncRNA MYLK-AS1 promotes the growth and invasion of HCC through the EGFR/HER2-ERK1/2 signaling pathway. However, the clinical significance of MYLK-AS1 in HCC still needs to be further determined. METHODS: Bioinformatic analysis was performed to determine the potential relationship among MYLK-AS1, miRNAs and mRNAs. A total of 156 samples of normal liver and paired HCC tissues from HCC patients were used to evaluate MYLK-AS1 expression by qRT-PCR. Human HCC cell lines were used to evaluate the colony formation, cell proliferation, migration, invasion, cell cycle and apoptosis after transfection of lentiviral short-hairpin RNAs (shRNAs) targeting MYLK-AS1 or MYLK-AS1 vectors. The competitive endogenous RNA (ceRNA) mechanism was clarified using fluorescence in situ hybridization (FISH), Western blotting, qPCR, RNA binding protein immunoprecipitation (RIP), and dual luciferase reporter analysis. RESULTS: MYLK-AS1 up-regulation was detected in the HCC tumor tissues and cell lines associated with the enhancement of the angiogenesis and tumor progression. The down-regulation of MYLK-AS1 reversed the effects on angiogenesis, proliferation, invasion and metastasis in the HCC cells and in vivo. MYLK-AS1 acted as ceRNA, capable of regulating the angiogenesis in HCC, while the microRNA miR-424-5p was the direct target of MYLK-AS1. Promoting the angiogenesis and the tumor proliferation, the complex MYLK-AS1/miR-424-5p activated the VEGFR-2 signaling through E2F7, whereas the specific targeting of E2F transcription factor 7 (E2F7) by miR-424-5p, was indicated by the mechanism studies. CONCLUSIONS: MYLK-AS1 and E2F7 are closely related to some malignant clinicopathological features and prognosis of HCC, thus the MYLK-AS1/ miR-424-5p/E2F7 signaling pathway might represent a promising treatment strategy to combat HCC.

Short-Step Adjustment and Proximal Compensatory Strategies Adopted by Stroke Survivors With Knee Extensor Spasticity for Obstacle Crossing.

Stroke survivors adopt cautious or compensatory strategies for safe and successful obstacle crossing. Although knee extensor spasticity is a common independent secondary sensorimotor disorder post-stroke, few studies have examined the step adjustment and compensatory strategies used by stroke survivors with knee extensor spasticity during obstacle crossing. This study aimed to compare the differences in the kinematics and kinetics during obstacle crossing between stroke survivors with and without knee extensor spasticity, and to identify knee extensor spasticity-related differences in step adjustment and compensatory strategies. Twenty stroke subjects were divided into a spasticity group [n = 11, modified Ashworth scale (MAS) ≥ 1] and a non-spasticity group (n = 9, MAS = 0), based on the MAS score of the knee extensor. Subjects were instructed to walk at a self-selected speed on a 10-m walkway and step over a 15 cm obstacle. A ten-camera 3D motion analysis system and two force plates were used to collect the kinematic and kinetic data. During the pre-obstacle phase, stroke survivors with knee extensor spasticity adopted a short-step strategy to approach the obstacle, while the subjects without spasticity used long-step strategy. During the affected limb swing phase, the spasticity group exhibited increased values that were significantly higher than those seen in the non-spasticity group for the following measurements: pelvic lateral tilt angle, trunk lateral tilt angle, medio-lateral distance between the ankle and ipsilateral hip joint, hip work contributions, the inclination angles between center of mass and center of pressure in anterior-posterior and medio-lateral directions. These results indicate that the combined movement of the pelvic, trunk lateral tilt, and hip abduction is an important compensatory strategy for successful obstacle crossing, but it sacrifices some balance in the sideways direction. During the post-obstacle phase, short-step and increase step width strategy were adopted to reestablish the walking pattern and balance control. These results reveal the step adjustment and compensatory strategies for obstacle crossing and also provide insight into the design of rehabilitation interventions for fall prevention in stroke survivors with knee extensor spasticity.

Voluntary exercise training attenuated the middle-aged maturity-induced cardiac apoptosis.

AIMS: Voluntary exercise training has cardioprotective effects in humans, but the underlying mechanism is unknown. This research was done to estimate the effect of voluntary exercise training to attenuate middle-aged maturity-induced cardiac apoptosis. MATERIALS AND METHODS: The study was designed to divide 64 male mice randomly into four groups, consisting of a 9-month sedentary pre-middle-aged group (9M), 15-month sedentary middle-aged group (15M), and two exercise groups using a voluntary wheel running respectively (9M+EX, 15M+EX). After 3 months, the condition of cardiac apoptosis in different groups was measured by HE dying, TUNEL and DAPI staining, and Western Blot analysis. KEY FINDINGS: TUNEL-positive cells were increased in 15M group compared with 9M group, while decreased in 9M+EX and 15M+EX groups compared with their control groups respectively. Protein levels of AIF, Endo G, TNF-α, TNFR1, TRAF2, TRADD, Fas, FasL, FADD, activated caspase 8, 3, 9, Bax/Bcl2, Bak/BclxL, and tBid were decreased in 9M+EX and 15M+EX groups compared with their control groups respectively. The protein levels of pBad/Bad, 14-3-3, IGF1, IGFR1, pPI3K/PI3K, and pAKT/AKT were more activated in the 9M+EX and 15M+EX groups than those in their control groups respectively. Significant differences were found between 9M group and 15M group for the protein levels of TRAF2, FADD, Bax/Bcl2, tBid and pAKT/AKT. SIGNIFICANCE: Voluntary exercise training as an important lifestyle modification may prevent cardiac widely dispersed apoptosis and enhance cardiac survival at middle-aged maturity.

Neuroprotective Effect of Rhodiola crenulata in D-Galactose-Induced Aging Model.

The medicinal plant Rhodiola crenulata grows at high altitudes in the Arctic and mountainous regions and is commonly used in phytotherapy in Eastern European and Asian countries. In the present study, we investigated the anti-apoptotic effect of Rhodiola crenulata and its neuroprotective mechanism of action in a rat model of D-galactose-induced aging. Two groups of twelve-week-old male Wistar rats received a daily injection of D-galactose (150mg/kg/day, i.p.) and orally administered Rhodiola crenulata (0, 248mg/kg/day) for eight weeks, while a control group received a saline injection (1ml/kg/day, i.p.). We examined apoptosis in the cortex and hippocampus of three groups of rats based on a terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL) positive assay. The expression levels of apoptotic and anti-apoptotic proteins in excised brains were analyzed by Western blotting. Our findings indicated that D-galactose caused marked neuronal apoptosis via activation of both extrinsic-dependent and mitochondrial-dependent apoptotic pathways. When compared to the control group, the protein levels of Fas receptor, Fas ligand, Fas-associated death domain (FADD), and activated caspase-8 (Fas-dependent apoptotic pathways), as well as those of t-Bid, Bax, cytochrome c, activated caspase-9, and activated caspase-3 (mitochondrial-dependent apoptotic pathways), were significantly increased in the D-galactose treated group. In addition, D-galactose impaired the phosphorylation of PI3K/Akt, an important survival signaling event in neurons. Rhodiola crenulata, however, protected against all these neurotoxicities in aging brains. The present study suggests that neuronal survival promoted by Rhodiola crenulata may be a potentially effective method to enhance the resistance of neurons to age-related disorders.

Antiapoptotic and mitochondrial biogenetic effects of exercise training on ovariectomized hypertensive rat hearts.

This study was to investigate the effects of exercise training on antiapoptotic pathways and mitochondrial biogenesis in ovariectomized hypertensive rats. Histopathological analysis, TUNEL assay, and Western blotting were performed on the excised hearts from female spontaneously hypertensive rats (SHR), which were divided into a sham-operated sedentary hypertensive (SHR-S), a sedentary hypertensive ovariectomized (SHR-O), and hypertensive ovariectomized rats that underwent treadmill exercise training (SHR-OT; 60 min/day, 5 days/wk) for 8 wk, along with normotensive Wistar Kyoto rats (WKY). When compared with the WKY group, the SHR-S group exhibited decreased protein levels of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial OPA-1 (mitochondrial biogenesis) and decreased further in the SHR-O group. The protein levels of p-PI3K, p-Akt, Bcl-2, Bcl-xL (prosurvival pathways), and the protein levels of PGC-1α and mitochondrial OPA1 (mitochondrial biogenesis) were increased in the SHR-OT group, but estrogen receptor (ER)α and ERß were not changed when compared with the SHR-O group. The protein levels of t-Bid, Bad, Bax, cytosolic cytochrome c, activated caspase 9, and activated caspase 3 (mitochondria-dependent apoptotic pathways), as well as Fas ligand, TNF-α, Fas receptors, Fas-associated death domain, activated caspase 8 (Fas receptor-dependent apoptotic pathways) were decreased in the SHR-OT group, when compared with the SHR-O group. Exercise training protection on the coexistence of hypertension and ovariectomy-induced cardiac mitochondria-dependent and Fas receptor-dependent apoptotic pathways by enhancing the Bcl2-related and mitochondrial biogenetic prosurvival pathways might provide a new therapeutic effect on cardiac protection in oophorectomized early postmenopausal hypertensive women. NEW & NOTEWORTHY Widely dispersed cardiac apoptosis was found in the coexistence of hypertension and ovariectomy. Exercise training on a treadmill could prevent ovariectomized hypertension-induced widely dispersed cardiac apoptosis via mitochondria-dependent apoptotic pathway (t-Bid, Bad, Bax, cytosolic cytochrome c, activated caspase 9, and activated caspase 3) and Fas receptor-dependent apoptotic pathway (Fas ligand, tumor necrosis factor-α, Fas receptors, Fas-associated death domain, activated caspase 8, and activated caspase 3) through enhancing the Bcl2-related (p-PI3K, p-Akt, Bcl-2, Bcl-xL) and mitochondrial biogenetic (PGC-1α and mitochondrial optic atrophy 1) prosurvival pathways.

Cross-talk among AFAP1-AS1, ACVR1 and microRNA-384 regulates the stemness of pancreatic cancer cells and tumorigenicity in nude mice.

BACKGROUND: Pancreatic cancer (PC) represents one of the most aggressive forms of cancer. The role of long non-coding RNAs (lncRNAs) has been highlighted in various malignancies including PC. The aim of the present study was to investigate the effects associated with actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) on the progression of PC and the underlying mechanism. METHODS: Microarray-based gene expression profiling of PC was performed to identify PC-related lncRNAs, after which the expression of AFAP1-AS1 and cancer stem cell (CSC) markers in PC tissues and cells were determined accordingly. The potential microRNA-384 (miR-384) capable of binding to AFAP1-AS1, in addition to its ability to regulate activin receptor A type I (ACVR1) were analyzed. In order to investigate the effect of the AFAP1-AS1/miR-384/ACVR1 axis on self-renewal ability, tumorigenicity, invasion, migration and stemness of PC cells, shRNA-AFAP1-AS1, miR-384 mimic and inhibitor were cloned into cells. RESULTS: High expression of AFAP1-AS1 and ACVR1 with low expression of miR-384 were detected in PC tissues. ACVR1 was determined to be down-regulated when miR-384 was overexpressed, while the inhibition of AFAP1-AS1 decreased its ability to binding competitively to miR-384, resulting in the down-regulation of ACVR1 and enhancing miR-384 expression, ultimately inhibiting the progression of PC. The knockdown of AFAP1-AS1 or overexpression of miR-384 was confirmed to impair PC cell self-renewal ability, tumorigenicity, invasion, migration and stemness. CONCLUSIONS: Taken together, AFAP1-AS1 functions as an endogenous RNA by competitively binding to miR-384 to regulate ACVR1, thus conferring inhibitory effects on PC cell stemness and tumorigenicity.

Combined effects of 17ß-estradiol and exercise training on cardiac apoptosis in ovariectomized rats.

BACKGROUND: The purpose of this study was to investigate the combined 17ß-estradiol (E2) and exercise training on cardiac pro-survival and anti-apoptotic pathways in ovariectomized rats. METHODS: Fifty-six female Sprague-Dawley rats were divided into a sham-operated (Sham), a bilaterally ovariectomized (OVX), an OVX treated with E2 (OVX-E2; 10µg/kg/day), and an OVX with E2 and treadmill exercise training (OVX-E2-EX; 60 min/day, 5 days/week) for 10 weeks. Following 10 weeks of exercise training, rat hearts were isolated for the evaluation of Histopathological analysis, TUNEL assay, and Western blotting. RESULTS: The protein levels of estrogen receptor α (ERα), estrogen receptor ß (ERß), insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), phospho-phosphatidylinositol 3-kinase (p-PI3K) (estrogen receptors/IGF-1-related survival pathway) were significantly increased in either the OVX-E2 or OVX-E2-EX group when compared with the OVX group. The protein levels of B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xL) and phosphorylated-Bad (p-Bad) (Bcl-2 family survival pathway) were significantly increased in the OVX-E2-EX group when compared with the OVX group. Only the p-Bad was significantly increased in the OVX-E2 group when compared with the OVX group. The protein levels of truncation of Bid (t-Bid), Bcl-2-associated death promotor (Bad), Bcl-2-associated X protein (Bax), Cytochrome c, caspases-9, and caspases-3 (mitochondria-dependent apoptotic pathway), as well as the protein levels of tumor necrosis factor-α (TNF-α), Fas ligand, Fas receptors, Fas-associated death domain (FADD), activated caspase-8 and activated caspase-3 (Fas receptor-dependent apoptotic pathway) were significantly decreased in either the OVX-E2 or OVX-E2-EX group when compared with the OVX group. Furthermore, when compared with the OVX-E2 group, the protein levels of ERß, IGF-1, IGF-1R, Bcl-2 and Bcl-xL were further enhanced in the OVX-E2-EX group as well as the protein levels of Cytochrome c, Fas receptors, FADD, activated caspase-8, activated caspase-9 and activated caspase-3 were further decreased in the OVX-EX-E2 group. CONCLUSIONS: Combined E2 and exercise training exhibited a positive effect of protection on ovariectomy-induced cardiac apoptosis by enhancing ERß-related survival pathways, which might provide a more effective therapeutic effect on cardiac protection in bilaterally oophorectomized or menopausal women than E2 treatment only.

Preparation of three-dimensional macroporous chitosan-gelatin B microspheres and HepG2-cell culture.

Chitosan-gelatin B microspheres with an open, interconnected, highly macroporous (100-200 µm) structure were prepared via a three-step protocol combining freeze-drying with an electrostatic and ionic cross-linking method. Saturated tripolyphosphate ethanol solution (85% ethanol) was chosen as the crosslinking agent to prevent destruction of the porous structure and to improve the biostability of the chitosan-gelatin B microspheres, with N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide/N-hydroxysuccinimide as a second crosslinking agent to react with gelatin A and fixed chitosan-gelatin B microspheres to attain improved biocompatibility. Water absorption of the three-dimensional macroporous chitosan-gelatin B microspheres (3D-P-CGMs) was 12.84, with a porosity of 85.45%. In vitro lysozyme degradation after 1, 3, 5, 7, 10, 14, and 21 days showed improved biodegradation in the 3D-P-CGMs. The morphology of human hepatoma cell lines (HepG2 cells) cultured on the 3D-P-CGMs was spherical, unlike that of cells cultured under traditional two-dimensional conditions. Scanning electron microscopy and paraffin sections were used to confirm the porous structure of the 3D-P-CGMs. HepG2 cells were able to migrate inside through the pore. Cell proliferation and levels of albumin and lactate dehydrogenase suggested that the 3D-P-CGMs could provide a larger specific surface area and an appropriate microenvironment for cell growth and survival. Hence, the 3D-P-CGMs are eminently suitable as macroporous scaffolds for cell cultures in tissue engineering and cell carrier studies. Copyright © 2014 John Wiley & Sons, Ltd.

Proinflammatory effects and molecular mechanisms of interleukin-17 in intestinal epithelial cell line HT-29.

AIM: To evaluate the proinflammatory effects and molecular mechanisms of interleukin (IL)-17 in intestinal epithelial cell line HT-29. METHODS: HT-29 cells were cultured with IL-17, tumor necrosis factor (TNF)-α, or the combination of both IL-17 and TNF-α. Real-time PCR and Western blot were used to measure the gene expression levels of neutrophil chemokines CXCL1, CXCL2, CXCL5, CXCL6, IL-8 and TH-17 cell chemokine CCL20, the phosphorylation levels of p38 and TNF-α, and the expression level of IL-8, after using the p38 inhibitor in HT-29 cells. The stable Act1 knockdown HT-29 cell line was established to further test the phosphorylation changes of p38, after using IL-17 and TNF-α. RESULTS: After HT-29 cells were cultured with IL-17 and TNF-α, the expression levels of neutrophil chemokines (CXCL1, CXCL2, CXCL5, CXCL6, IL-8) and Th17 chemokine (CCL20) significantly improved (24.96 ± 2.53, 28.47 ± 2.87, 38.08 ± 2.72, 33.47 ± 2.41, 31.7 ± 2.38, 44.37 ± 2.73, respectively), and the differences were all statistically significant (P < 0.01). Western blot results showed that IL-17 obviously enhanced the phosphorylation level of p38, which was induced by TNF-α. Compared with the control group, the expression level of IL-8 significantly declined (9.47 ± 1.36 vs 3.06 ± 0.67, P < 0.01) when TH-29 cells were cultured with IL-17 and TNF-α. p38 inhibition assay showed that the p38 pathway played an essential role in the inflammatory response induced by IL-17. p38 phosphorylation levels could not be changed after using IL-17 and TNF-α in the stable Act1 knockdown HT-29 cell line. CONCLUSION: IL-17 significantly promoted the gene expression levels of TNF-α-induced neutrophil chemokines and Th17 cell chemokine. It is obvious that IL-17 and TNF-α have synergistic effects on p38.

Hepatocyte differentiation of mesenchymal stem cells.

BACKGROUND: Liver cell transplantation and bioartificial liver may provide metabolic support of liver function temporary and are prospective treatments for patients with liver failure. Mesenchymal stem cells (MSCs) are expected to be an ideal cell source for transplantation or liver tissue engineering, however the hepatic differentiation of MSCs is still insufficient for clinical application. DATA SOURCES: A PubMed search on "mesenchymal stem cells", "liver cell" and "hepatocyte differentiation" was performed on the topic, and the relevant articles published in the past ten years were reviewed. RESULTS: Hepatocyte-like cells differentiated from MSCs are a promising cell source for liver regeneration or tissue engineering. Although it is still a matter of debate as to whether MSC-derived hepatocytes may efficiently repopulate a host liver to provide adequate functional substitution, the majority of animal studies support that MSCs can become key players in liver-directed regenerative medicine. However the clinical application of human stem cells in the treatment of liver diseases is still in its infancy. CONCLUSIONS: Future studies are required to improve the efficacy and consistency of hepatic differentiation from MSCs. It is necessary to better understand the mechanism to achieve transdifferentiation with high efficiency. More clinical trials are warranted to prove their efficacy in the management of patients with liver failure.

Preparation and characterization of chitosan porous microcarriers for hepatocyte culture.

BACKGROUND: The bioartificial liver (BAL) is considered a possible alternative method for treating liver failure. The core of the BAL system is culturing liver cells in vitro with high density and activity. Microcarrier culture is a mode of high-density culture. We set out to prepare a novel porous microcarrier to improve the activity of liver cells in vitro. METHODS: Chitosan was used to prepare a novel porous spherical microcarrier with interconnected structure. The chitosan porous microcarriers (CPMs) were modified with gelatin to improve their biocompatibility. CPMs were co-cultured with liver cells, HL-7702 (L-02), to evaluate their effect on cell culture. RESULTS: The average size of the CPMs was about 400 µm in diameter and their apertures were less than 30 µm. The pores of the microcarrier were interconnected. After fixation by sodium tripolyphosphate, the structure of the first freeze-dried CPMs was stable. To further improve the biocompatibility, the surface of CPMs was modified with gelatin through chemical crosslinking (GM-CPMs). Comparing the proliferation curves of L-02 cells cultured on simple CPMs, GM-CPMs and tissue culture polystyrene (TCPS, a mode of planar cell culture), the proliferation rates were similar in the first 5 days and the cells proliferated until day 8 in culture with microcarriers. The OD value of liver cells cultured on GM-CPMs was 1.97-fold higher than that on TCPS culture at day 8. Levels of urea and albumin in supernatants of cells cultured on GM-CPMs increased steadily for 8 days, and were clearly higher than those of cells cultured on TCPS (P<0.05). CONCLUSIONS: The novel CPMs were promising microcarriers for hepatocyte culture and the GM-CPM seemed better. Porous microcarrier culture was beneficial for hepatocyte function and activity.