Costal Cartilage Graft Repair Osteochondral Defect in a Mouse Model.

OBJECTIVE: Osteochondral defects develop into osteoarthritis without intervention. Costal cartilage can be utilized as an alternative source for repairing osteochondral defect. Our previous clinical study has shown the successful osteochondral repair by costal cartilage graft with integration into host bone bed. In this study, we investigate how cartilaginous graft adapt to osteochondral environment and the mechanism of bone-cartilage interface formation. DESIGN: Costal cartilage grafting was performed in C57BL/6J mice and full-thickness osteochondral defect was made as control. 3D optical profiles and micro-CT were applied to evaluate the reconstruction of articular cartilage surface and subchondral bone as well as gait analysis to evaluate articular function. Histological staining was performed at 2, 4, and 8 weeks after surgery. Moreover, costal cartilage from transgenic mice with fluorescent markers were transplanted into wild-type mice to observe the in vivo changes of costal chondrocytes. RESULTS: At 8 weeks after surgery, 3D optical profiles and micro-CT showed that in the graft group, the articular surface and subchondral bone were well preserved. Gait analysis and International Cartilage Repair Society (ICRS) score evaluation showed a good recovery of joint function and histological repair in the graft group. Safranin O staining showed the gradual integration of graft and host tissue. Costal cartilage from transgenic mice with fluorescent markers showed that donor-derived costal chondrocytes turned into osteocytes in the subchondral area of host femur. CONCLUSION: Costal cartilage grafting shows both functional and histological repair of osteochondral defect in mice. Graft-derived costal chondrocytes differentiate into osteocytes and contribute to endochondral ossification.

Inhibitory effect of tumor necrosis factor-α on the basolateral Kir4.1/Kir5.1 channels in the thick ascending limb during diabetes.

Diabetic nephropathy is a major contributor to the morbidity and mortality of patients with diabetes. TNF-α expression is elevated during diabetes and is implicated in the pathogenesis of diabetic nephropathy; however, its underlying molecular mechanisms remain unclear. The present study aimed to investigate the effect and molecular mechanism of TNF-α on the basolateral inwardly rectifying potassium (Kir)4.1/Kir5.1 channels in the thick ascending limb (TAL) of rat kidneys using western blotting and the patch clamp technique to provide a theoretical basis for the cause of the decrease in kidney concentrating capacity during diabetes. The results demonstrated that urinary TNF-α excretion and protein TNF-α expression in the TAL increased and basolateral Kir4.1/Kir5.1 channel activity decreased during diabetes; however, diabetic rats exhibited amelioration of Kir4.1/Kir5.1 activity with a soluble TNF-α antagonist, TNF receptor fusion protein (TNFR:Fc). These results suggested that TNF-α inhibited the activity of the basolateral Kir4.1/Kir5.1 channel in the TAL of rat kidneys during diabetes. In addition, the protein expression levels of phospholipase A2 (PLA2) and cyclooxygenase-2 (COX2) increased in diabetic rats, the effects of which deceased following treatment with TNFR:Fc compared with the diabetic group. Furthermore, an agonist of PLA2 (melittin) and COX2 production [prostaglandin E2 (PGE2)] inhibited the basolateral Kir4.1/Kir5.1 channels. Taken together, the results of the present study suggested that the inhibitory effect of TNF-α on the basolateral Kir4.1/Kir5.1 channels in the TAL during diabetes is mediated by the PLA2/COX2/PGE2 signaling pathway.

CXCL3 overexpression promotes the tumorigenic potential of uterine cervical cancer cells via the MAPK/ERK pathway.

CXCL3 belongs to the CXC-type chemokine family and is known to play a multifaceted role in various human malignancies. While its clinical significance and mechanisms of action in uterine cervical cancer (UCC) remain unclear. This investigation demonstrated that the UCC cell line HeLa expressed CXCL3, and strong expression of CXCL3 was detected in UCC tissues relative to nontumor tissues. In addition, CXCL3 expression was strongly correlated with CXCL5 expression in UCC tissues. In vitro, HeLa cells overexpressing CXCL3, HeLa cells treated with exogenous CXCL3 or treated with conditioned medium from WPMY cells overexpressing CXCL3, exhibited enhanced proliferation and migration activities. In agreement with these findings, CXCL3 overexpression was also associated with the generation of HeLa cell tumor xenografts in athymic nude mice. Subsequent mechanistic studies demonstrated that CXCL3 overexpressing influenced the expression of extracellular signal-regulated kinase (ERK) signaling pathway associated genes, including ERK1/2, Bcl-2, and Bax, whereas the CXCL3-induced proliferation and migration effects were attenuated by exogenous administration of the ERK1/2 blocker PD98059. The data of the current investigation support that CXCL3 appears to hold promise as a potential tumor marker and interference target for UCC.

Acute stimulatory effect of tumor necrosis factor on the basolateral 50 pS K channels in the thick ascending limb of the rat kidney.

The aim of the present study was to investigate the acute effect and mechanism of tumor necrosis factor (TNF) on basolateral 50 pS K channels in the thick ascending limb (TAL) of the rat kidney. The TAL tubules were isolated from the rat kidney, and the activity of the 50 pS K channels was recorded using the patch­clamp technique. The results indicated that the application of TNF (10 nM) significantly activated the 50 pS K channels and the TNF effect was concentration­dependent. Inhibition of protein kinase A, phospholipase A2 and protein tyrosine kinase using pathway inhibitors (H89, AACOCF3 and Herbimycin A, respectively) did not abolish the stimulatory effect of TNF, indicating that none of these pathways mediated the TNF effect. By contrast, the phenylarsine oxide inhibitor against protein tyrosine phosphatase (PTP) decreased the activity of the 50 pS K channels and blocked the stimulatory effect of TNF on these channels. Furthermore, western blot analysis demonstrated that the application of TNF (10 nM) in the TAL increased the phosphorylation of PTP, an indication of PTP activity stimulation. Thus, it was concluded that the acute application of TNF may stimulate the basolateral 50 pS K channel in the TAL and the stimulatory effect of TNF may be mediated by the PTP­dependent pathway.

High C-X-C motif chemokine 5 expression is associated with malignant phenotypes of prostate cancer cells via autocrine and paracrine pathways.

The present study aimed to examine the effects and mechanisms of exogenous C-X-C motif chemokine 5 (CXCL5) and lentiviral CXCL5 overexpression on the regulation of malignant behaviors of prostate cancer cells in vitro and in a nude mouse xenograft model. The expression levels of CXCL5 and a number of tumor-related genes were assessed by using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), western blotting, ELISA, or immunohistochemistry in normal and cancerous prostate cells and tissues. Cell proliferation, colony formation, and Transwell assays were performed to determine the effects of exogenous, autocrine, and paracrine CXCL5 on prostate cancer cell proliferative and migratory capacity. The results indicated that CXCL5 expression was upregulated in PC­3 and DU145 prostate cancer cells, in WPMY­1 normal prostate stromal cells, and in RWPE­1 prostate epithelial cells, as well as in prostate cancer tissue specimens. Exogenous CXCL5 exposure resulted in increase in prostate cancer cell proliferation, colony formation, and migration. In cells transfected with a CXCL5 overexpression vector, in cells cultured in conditioned medium from CXCL5-overexpressing WPMY cells, and in cells co-cultured with CXCL5­OE WPMY cells prostate cancer cell malignant phenotypes were induced in an autocrine/paracrine fashion in vitro; similar results were observed in nude mouse xenografts. CXCL5 overexpression also regulated expression of tumor-related genes, including BAX, N-Myc downstream-regulated gene 3, extracellular signal-regulated kinase 1/2, C-X-C chemokine receptor type 2, interleukin 18, Bcl­2, and caspase­3. These data demonstrated that CXCL5 expression was upregulated in prostate cancer tissues and that exogenous CXCL5 protein exposure or CXCL5 overexpression promoted malignant phenotypes of prostate cancer cells in vitro and in vivo.

CXCL5 as an autocrine or paracrine cytokine is associated with proliferation and migration of hepatoblastoma HepG2 cells.

C-X-C motif chemokine ligand 5 (CXCL5) is a CXC-type chemokine that is a crucial inflammatory mediator and a powerful attractant for granulocytic immune cells. Increasing evidence has indicated that CXCL5 is involved in the tumorigenesis of various malignancies. The present investigation demonstrated that CXCL5 was expressed in both hepatoblastoma HepG2 cells and liver stellate LX-2 cells, and CXCL5's receptor C-X-C chemokine receptor type 2 (CXCR2) was expressed in HepG2 cells by reverse transcription-polymerase chain reaction (RT-PCR), western blotting and ELISA assays. Cell counting kit-8, colony formation and Transwell assays revealed that exogenous CXCL5 expression efficiently promoted proliferation, colony formation and migration of HepG2 cells. To explore the autocrine and paracrine roles of CXCL5 in the oncogenic potential of HepG2 cells, HepG2 cells overexpressing CXCL5 and LX-2 cells overexpressing CXCL5 were successfully constructed by gene transfection. Similarly, overexpression of CXCL5 in HepG2 also enhanced proliferation, colony formation and migration of HepG2 cells. Furthermore, the condition medium of LX-2 cells overexpressing CXCL5 affected the proliferation and migration of HepG2 cells. RT-PCR and western blotting assays were also conducted to explore whether overexpression of CXCL5 in HepG2 modulated the expression of genes. The results revealed that overexpression of CXCL5 regulated the expression of several genes, including N-myc downregulated gene 3,w B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein, P53, vascular endothelial growth factor, interleukin (IL)-18, IL-1ß and cystathionine-γ-lyase. In conclusion, the present findings indicate that CXCL5/CXCR2 axis contributes to the oncogenic potential of hepatoblastoma via autocrine or paracrine pathways by regulating expression of genes associated with the progression of carcinoma.

Effects of adenosine stimulation on the mRNA expression of CLCNKB in the basolateral medullary thick ascending limb of the rat kidney.

Adenosine is a molecule produced by several organs within the body, including the kidneys, where it acts as an autoregulatory factor. It mediates ion transport in several nephron segments, including the proximal tubule and the thick ascending limb (TAL). Ion transport is dictated in part by anionic chloride channels, which regulate crucial kidney functions, including the reabsorption of Na+ and Cl­, urine concentration, and establishing and maintaining the corticomedullary osmotic gradient. The present study investigated the effects of adenosine on the mRNA expression of chloride voltage­gated channel Kb (CLCNKB), a candidate gene involved in hypertension, which encodes for the ClC­Kb channel. Medullary thick ascending limb (mTAL) tubules were isolated from the rat kidney, and primary cultures of mTAL cells from the mTAL tubules were established. The cells were treated with adenosine and the mRNA expression of CLCNKB was detected by reverse transcription­quantitative polymerase chain reaction. The cells were also treated with pathways inhibitors (H8 and AACOCF3), and the protein expression of cyclic adenosine 3',5'­monophosphate (cAMP)­protein kinase A (PKA) and phospholipase A2 (PLA2) by were analyzed by western blotting. The findings indicated that adenosine increased the mRNA expression of CLCNKB in primary cultures of medullary TAL cells, and this stimulatory effect was regulated by the cAMP­PKA and PLA2­arachidonic acid (AA) pathways. The present study showed that adenosine affected the mRNA expression of CLCNKB, initially through the cAMP­PKA pathway and then the PLA2­AA pathway.

Overexpression of CXCL3 can enhance the oncogenic potential of prostate cancer.

PURPOSE: CXCL3 and its receptor CXCR2 were considered to play particularly important roles in the progression of malignancies. However, the investigations about CXCL3/CXCR2 axis in prostate cancer have been poorly involved. Herein we firstly reported our studies on the expression and biological roles of CXCL3 and CXCR2 in prostate cancer. METHODS: Expression levels of CXCL3 and CXCR2 in prostate cancer cell lines (PC-3, DU145 and LNCaP), immortalized prostate stromal cell line (WPMY-1) and immortalized prostate epithelial cell line (RWPE-1) were investigated by RT-PCR, ELISA and western blot, whereas expression levels of CXCL3 in a prostate tissue microarray were detected by immunohistochemistry. Cell counting kit-8 and transwell assays were, respectively, utilized to determine the effects of exogenous CXCL3 on the cell proliferation and migration. We further examined whether CXCL3 could regulate the expression of genes correlated with prostate tumorigenesis by RT- PCR. RESULTS: Elevated expression of CXCR2 was detected in DU145, LNCaP and RWPE-1. Moreover, high-level CXCL3 can be secreted by PC-3 and RWPE-1, and CXCL3 protein expression level in tissue microarray is concordant with prostate cancer metastasis. Exogenous CXCL3 does not contribute to proliferation, but has a significant effect on migration of prostate cancer cells and RWPE-1. Finally, our data showed that exogenous CXCL3 can regulate the expression of genes including ERK, TP73, NUMB, BAX and NDRG3. CONCLUSION: Our findings suggest that CXCL3 and its receptor CXCR2 are overexpressed in prostate cancer cells, prostate epithelial cells and prostate cancer tissues, which may play multiple roles in prostate cancer progression and metastasis.

Effects of platycodins on liver complications of type 2 diabetes.

The aim of the current study was to investigate the therapeutic effects and mechanism of platycodin in liver complications of type 2 diabetes. All rats were randomly divided into two groups: The control group (normal diet) and the model group (a high­fat and high­sugar diet). The model group was injected with 2% streptozocin (25 mg/kg body weight) through the tail vein following 4 weeks of dieting. After a total of 8 weeks of dieting, fasting blood glucose (FBG) and liver function were examined. The high­fat and high­sugar diet was continued in the successful model rats, which were randomly divided into four groups and treated with the following doses of platycodins: The untreated, and 50, 100 and 200 mg/kg body weight/day groups. Platycodins treatment lasted for 12 weeks. Platycodins treatment at a dose of 200 mg/kg body weight/day reduced the FBG, glutamate pyruvate transaminase (GPT), glutamic oxalacetic transaminase, triglycerides, total cholesterol (TC), low­density lipoprotein (LDL) and liver index levels compared with the untreated group (P<0.05), while the high­density lipoprotein levels increased (P<0.05). Furthermore, FBG, GPT, TC and LDL levels were returned to the normal level. This dose also increased the expression of BMP­9 mRNA and BMP­9 protein, and reduced the expression of Smad­4 mRNA and Smad­4 protein. These findings indicate that platycodins can rectify disorders of blood glucose and lipid metabolism, improve liver index and protect liver function in liver complications of type 2 diabetes. The current study suggests that this therapeutic effect is mediated through the BMP­9/Smad­4 pathway.

Involvement of protein kinase A activation and phospholipase A(2) inhibition in the adenosine-activated basolateral 50 pS K(+) channels in the thick ascending limb of the rat kidney.

The present study was designed to investigate the role of protein kinase A (PKA) and phospholipase A(2) (PLA(2)) in the stimulating effect of adenosine on the basolateral 50 pS K(+) channels in the thick ascending limb (TAL) of the rat kidney. Under the anatomic microscope, the TAL was dissected. The current of 50 pS K(+) channels were recorded by patch clamp technology. The protein expression of phosphorylated PKA and phosphorylated PLA(2) were examined by Western blot. The results showed that cyclohexyladenosine (CHA), an analog of adenosine, increased the 50 pS K(+) channel activity (P < 0.05). In the presence of H8, an antagonist of PKA, CHA did not affect the 50 pS K(+) channel activity. In the presence of AACOCF3 (an antagonist of PLA(2)), CHA did not further increase the 50 pS K(+) channel activity. CHA increased phosphorylation level of PKA, whereas inhibited phosphorylation of PLA(2) in the TAL of the rat kidney (P < 0.01). Furthermore, after blocking the PLA(2) with AACOCF3, CHA still increased the expression of phosphorylated PKA. On the contrary, CHA did not obviously change the expression of phosphorylated PLA(2) after H8 pretreatment. The results suggest that the stimulation of basolateral 50 pS K(+) channels by CHA is mediated by the activation of PKA followed by the inhibition of PLA(2) in the TAL of the rat kidney.

Stimulation of A(2a) adenosine receptor abolishes the inhibitory effect of arachidonic acid on the basolateral 50-pS K channel in the thick ascending limb.

The basolateral 50-pS K channels are stimulated by a cAMP-dependent pathway and inhibited by cytochrome P-450-omega-hydroxylase-dependent metabolism of arachidonic acid (AA) in the rat thick ascending limb (TAL). We now used the patch-clamp technique to examine whether stimulation of adenosine A(2a) receptor modulates the inhibitory effect of AA on the basolateral 50-pS K channels in the medullary TAL. Stimulation of adenosine A(2a) receptor with CGS-21680 or inhibition of phospholipase A2 (PLA2) with AACOCF3 increased the 50-pS K channel activity in the TAL. Western blot demonstrated that application of CGS-21680 decreased the phosphorylation of PLA(2) at serine residue 505, an indication of inhibiting PLA2 activity. In the presence of CGS-21680, inhibition of PLA2 had no further effect on the basolateral 50-pS K channels. The possibility that CGS-21680-induced stimulation of the basolateral 50-pS K channels was partially achieved by inhibition of PLA2 in the TAL was also supported by the observation that CGS-21680 had no additional effect in the presence of AACOCF3. Moreover, stimulation of adenosine A(2a) receptor with CGS-21680 also abolished the inhibitory effect of AA and 20-hydroxyeicosatetraenoic acid (20-HETE) on the 50-pS K channels. The effect of CGS-21680 on AA and 20-HETE-mediated inhibition of the 50-pS K channels was mediated by cAMP because application of membrane-permeable cAMP analog, dibutyryl-cAMP, not only increased the 50-pS K channel activity but also abolished the inhibitory effect of AA and 20-HETE. We conclude that stimulation of adenosine A(2a) receptor increased the 50-pS K channel activity in the TAL, an effect that is achieved by suppression of PLA2 activity and 20-HETE-induced inhibition.