Erythropoietin （EPO） is known as a classical hematopoietic growth factor, which has been used to treat anemia caused by different reasons. In recent years, EPO＇s non-hematopoietic biological effects have gradually become a focus. Among these effects, EPO＇s tissue protection is most attractive and EPO has been proved to protect many different tissues and organs. Myocardial protection has always been the important and key topic in the field of cardiovascular diseases. Reports about EPO＇s myocardial protective effects have been published in the recent two years, which direct the research about myocardial protection with new ideas. In this article, the discoveries and unsolved problems associated with EPO＇s myocardial protection were reviewed.
Objective To investigate the effect of exogenous erythropoietin (EPO) on the denervated muscle atrophy. Methods Twenty-four SD male rats, weighting 200-220 g were made the models of denervated gastrocnemius muscle after sciatic nerves were transected under the piriform muscle at the right lower leg, and were randomly divided into two groups (n=12). rhEPO (2 500 U/kg) was injected daily into the denervated gastrocnemius muscle in EPO group, and normal sal ine was injected into the denervated gastrocnemius muscle in control group. To observe the general state of health of the experimental animal, the muscle wet weight, the muscle cell diameter, the cross section area, the protein amount, thepercentage of the apoptotic muscle cells, and the Na+-K+-ATPase and Ca2+-ATPase activities were measured 2 and 4 weeks after operation. Results All experimental animals were survived during experiment without cut infection, and all animals could walk with pull ing the right knee. At 4 weeks after operation, 7 cases showed ulcer in the right heel, inculding 5 in the control group and 2 in the EPO group. At 2 and 4 weeks after operation, the muscle wet weight in EPO group was (885.59 ± 112.35) and (697.62 ± 94.74) g, respectively; in control group, it was (760.63 ± 109.05) and (458.71 ± 58.76) g, respectively; indicating significant differences between two groups (P lt; 0.01). The protein amount in EPO group was (77.37 ± 5.24) and (66.37 ± 4.87) mg/mL, respectivly；in control group, it was (65.39 ± 4.97) and (54.62 ± 6.32) mg/mL；indicating significant differences between two groups (P lt; 0.01). At 2 and 4 weeks after operation, the myofibrillar shapes were nearly normal in EPO group while there were muscle fiber atrophy, some collapse and obviously hyperblastosis between muscle bundle. There were significant differences in the muscle cell diameter and the cross section between two groups (P lt; 0.01). However, the percentage of the apoptotic muscle cells was 11.80% ± 1.74% and 28.47% ± 1.81% in control group, respectively, which was significantly smaller than that in EPO group (21.48% ± 2.21% and 55.89% ± 2.88%, P lt; 0.01). At 2 and 4 weeks after operation, Na+-K+-ATPaseand Ca2+-ATPase activities in EPO group were higher than those in control group (P lt; 0.01). Conclusion EPO can delay the denervated muscle atrophy.
Objective To investigate the protective effect of recombinant erythropoietin (EPO) on the photoreceptor cells in rat with retinal detachment (RD).Methods One hundred and sixtytwo normal male rats were randomly divided into normal control (NC) group, RD model group, RD+phosphate buffer solution （RD+PBS） group, RD+EPO 100 ng group, RD+EPO 200 ng group and RD+EPO 400 ng group. Three days after RD, activated caspase3 and bclXL were detected by Western blot and/or immunofluorescence, and apoptosis were measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick-end labeling（TUNEL）. Fourteen and 28 days and two months after RD, the outer nuclear layer （ONL）thickness was measured by histopathologic method.Results Western bolt indicated that the protein level of activated caspase-3 and bcl-XL between six groups were statistically significant（F=35.96, 30.75；P＜0.01）. The number of TUNEL positive cells and activated caspase-3 positive cells are consistent with each other in different groups. Fourteen days and two months after RD，the differences of ONL thickness between six groups were statistically significant（F=21.52，96.25；P＜0.01）.Conclusion Supplement of EPO after RD can alleviate apoptosis by inhibiting of the caspase-3 activity and increasing the expression of bcl-XL,thus exerts protective effect on photoreceptor cells.
Objective To observe the expression of erythropoietin (EPO) and EPO receptor (EPOR) in detached retina in rat model. Methods Fourty-eight male SD rats were randomly divided into control group and retinal detachment (RD) groups (1 hour, 3, 6, 12, 24, 48, and 72 hours group) with 6 rats (12 eyes) in each group. 1.4% hyaluronic acid was slowly injected into the subretinal space to induce the detachment of the upper retina to set up the RD model. The expression levels of mRNA and protein of EPO and EPOR were measured by RT-PCR and western-blotting analysis. Meanwhile, the locations of EPO and EPOR in retina were checked by immunohistochemistry. Results Both of the mRNA and protein levels of EPO and EPOR increased after RD, and reached the peak at the 48th hour after RD. The mRNA levels of EPO and EPOR were significantly higher in the 6 and 12 hours group than that in the control group（Plt;0.05）. The protein levels of EPO and EPOR were significantly higher in 3 hours group than that in the control group（Plt;0.05）. Immunohistochemistry indicated weak expression of EPO from ganglion cell layer to inner and outer segment of photoreceptor cells, and b expression in the corresponding location was found 48 hours after RD. Expression of EPOR from ganglion cell layer to inner segment of photoreceptor cells in the normal retina was detected, and b expression in the corresponding location was found 48 hours after RD. Conclusion The expression of EPO and EPOR in retina increases gradually after RD, and reaches the peak at the 48th hour; most of the layers of neural retina can express EPO and EPOR.
Objective To investigate the protective effect and mechanism of erythropoietin (EPO) on injury of human retinal pigment epithelial (hRPE) cell induced by hydrogen peroxide (H2O2). Methods Take subcultured hFRPE cells as study target. They were treated with 800 mu;mol/L of H2O2 for 3 hours to establish the cell injury model. The cultured cells were divided into three groups：control group, simply injury group and therapeutic group which again divided into 10 IU/ml, 20 IU/ml, 40 IU/ml,60 IU/ml subgroups according to the concentration of recombinant human erythropoietin(rhEPO). NF-kappa;B was measured by immunohistochemistry. The content of Malondialdehyde(MDA) which was the product of cellular lipid peroxidation and the releasing rate of lactate dehydrogenase（LDH）were estimated by chromatometry. Results H2O2 could elevate the level of MDA and the releasing rate of LDH, compared simply injury group with control group, the differences were significant.(tLDH＝29.746,tMDA＝20.426,Plt;0.05); Compared all of therapeutics groups with simply injury group, the releasing rate of MAD and LDH were decreased obviously, the differences were significant.（LDH t10IU＝5.770，t20IU＝12.774，t40IU＝19.818，t60IU＝24.833，Plt;0.05；MDA t10IU＝5.345，t20IU＝10.278，t40IU＝18.571，t60IU＝20.247，Plt;0.05）; The correlative analysis results of each therapeutic subgroup were: ①the concentration of rhEPO had negative correlation with the relation rate of LDH and the content of MDA(r=-0.976，P=0.024； r=-0.968，P=0.032) ; ②the concentration of rhEPO had positive correlation with the nuclear translative rate of NF-kappa;B(r=0.998，P=0.002); ③the nuclear translative rate of NF-kappa;B had negative correlation with the content of MDA(r=－0.954，P=0.046). Conclusion EPO can protect hFRPE cells from the injury of H2O2, the mechanism may be related to the activation of NF-kappa;B.
Objective To assess the protective effect of recombinant human erythropoietin (EPO) on human retinal pigment epithelial (RPE) cells injured by light. Methods Cultured human RPE cells were exposed to light for 12 hours, and the culture was stopped 24 hours later. The 3(4,5dimethylthiazole2y1)2,5diphenyl tetrazolium bromide (MTT) cell viability assay and annexin V flunorescein isothiocyanate/propidium iodium labeling and flow cytometry were used to assess the effects of EPO with different concentration on the cellular viability and apoptosis of human RPE cells. The protective effect and mechanism of EPO on RPE cells injured by light was detected by adding AG490. Results EPO, especially with the concentration of 40 IU/ml, obviously increased the cellular viability of RPE cells and apparently decrease the cellular apoptosis induced by light injury. After adding AG490, the effects of EPO on cellular viability and apoptosis were inhibited. Conclusion It is suggested that EPO can protect the human RPE cells from lightinduced injures, and its protective mechanism works after the combination of EPO and its receptor.
Objective To investigate the effect of hypoxia on expressions of erythropoietin（EPO）mRNA and protein in retinal Muuml;ller cells cultured in vitro. Methods Retina tissues from the new-born Wistar rats were dissected into cell suspension after digested by pancreatin.Muuml;ller cells were separated and purified by mechanical concussion and blowing and striking method.The expression of EPO mRNA and protein under the condition of hypoxia was detected by semi-quantitative reverse transcriptase（RT）-polymerase chain reaction（PCR）and immunocytochemical method. Results Retinal Muuml;ller cells were cultured successfully，95% of which were positively stained by glial fibrillary acidic protein（GFAP）.Positively stained EPO protein was located in the cytoplasm and protuberance.The expression of EPO mRNA and protein was faint in the normal retinal Muuml;ller cells，but increased obviously and time-dependently after hypoxia. Conclusion Expression of EPO mRNA and protein increases in Muuml;ller cells after hypoxia，which may be one of the protective factors for the nerves in anoxic retinopathy. (Chin J Ocul Fundus Dis, 2006, 22: 196-199)
Objective To assess the effectiveness and safety of erythropoietin (EPO) for cancer-related malignant anemia without radiotherapy or chemotherapy. Methods Randomized controlled trials (RCTs) or quasi-randomized controlled trials (quasi-RCTs) involving erythropoietin in the treatment of cancer-related malignant anemia were searched and identified from PubMed (1966 to Sept. 2009), EMBASE (1974 to Sept. 2009), The Cochrane Library (Issue 3, 2009), CBM (1978 to Sept. 2009), CNKI (1994 to Sept. 2009), VIP (1989 to Sept. 2009). We also handsearched relevant journals. Data were extracted and evaluated by two reviewers independently with specially designed extraction form. We evaluated the quality of the included studies by the Cochrane Handbook 5.0 recommend standard and analyzed data by Cochrane Collaboration’s RevMan 5.0. Results We included twelve trials. The quality of the included studies was poor. The grade of ten studies was B, and the grade of two studies was C. Meta-analyses showed that there were significant differences between erythropoietin and blank in volume of blood transfusion [SMD= –0.66, 95%CI (–1.14, –0.17), P=0.008], number need to transfusion [OR=0.60, 95%CI (0.39, 0.92), P=0.02], and the change of hemoglobin after two-week therapy [SMD=2.40, 95%CI (0.29, 4.52), P=0.03]. Conclusion The current evidence shows that EPO significantly benefits cancer-related malignant anemia. Well-designed RCTs with a larger sample size, longer intervention and follow-up periods are still needed.
ObjectiveTo investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9 (BMP-9) and erythropoietin (EPO) genes co-transfection on osteogenic differentiation of adipose-derived stem cells (ADSCs) in vitro. MethodsThe inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups: normal cells (group A), empty plasmid control group (group B), BMP-9 or EPO recombinant adenovirus transfected cells (groups C and D), BMP-9 and EPO recombinant adenovirus co-transfected cells (group E). The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase (ALP) activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin (OPN) and osteocalcin (OCN) at 3 weeks. ResultsAt 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot (P < 0.05). The ALP activity significantly increased in group E when compared with that in the other groups at 3, 7, and 14 days after osteogenic induction (P < 0.05); the number of calcium nodules in group E was significantly more than that in the other groups (P < 0.05). Real-time fluorescence quantitative PCR showed that OPN and OCN genes expressions were significantly higher in group E than other groups (P < 0.05), and in groups C and D than groups A and B (P < 0.05). ConclusionRecombinant adenovirus-mediated BMP-9 and EPO genes can transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the expressions of osteoblast-related genes and protein than non-transfected and single gene transfection.