Effects of Coriaria sinica Maxim’s extract on burn wound healing and scar hyperplasia in rats based on ILK signaling pathway

AIM: To explore the mechanism of Coriaria sinica Maxim’s extract (CSME) promoting burn wound healing in the early stage and inhibiting excessive scar hyperplasia in the later stage, based on the signaling pathways, such as transforming growth factor-β₁ (TGF-β₁) regulated by integrin-linked kinase (ILK) and ILK regulated by PI3K/AKT. METHODS: Female SD rats (n=180; 180~200 g) were randomly divided into 6 groups: normal control (NC) group, vaseline (VL) group, silver sulfadiazine (SS) group and low-, medium- and high-dose of CSME (CSME-L, CSME-M and CSME-H) groups, with 30 rats in each group. Except for the rats in NC group, VL, SS, and 3 doses of CSME were applied to the wound surface of the rats in the corresponding groups every day after II° burn model was made on their waist-back in the condition of chloral hydrate anesthesia. To calculate the healing rate (HR), 10 rats in each experimental group were randomly selected to remove their wound skin for observing the pathologic change, detecting the expression of related proteins by Western blot and RT-qPCR, and checking the collagen shrinkage (SK) by fibroblast culture at the 7th, 14th, and 21st days. RESULTS: The expression of ILK, fibronectin (FN), TGF-β₁, α-smooth muscle actin (α-SMA) and integrin-β₁ (ITG-β₁) at protein and mRNA levels in wound skin of CSME groups was stronger than that in VL group and SS group at the 7th day in a dose-dependent manner, but weaker than that in VL group and SS group at the 21st day (P<0.05). Meanwhile, the protein and mRNA expression of collagen type I (Col I) in CSME groups was stronger than that in VL group and SS group from the 7th day to the 14th day, but weaker than that in VL group and SS group at the 21st day in a dose-dependent manner (P<0.05). However, the protein and mRNA expression of collagen type III (Col III) in CSME groups was weaker than that in VL group and SS group from the 7th day to the 14th day, but stronger than that in VL group and SS group at the 21st day in a dose-dependent manner (P<0.05). The SK of fibroblasts in VL group and SS group was increased continuously over time and reached its peak at 96 h. SK in CSME groups was only higher than that in VL group and SS group at 24 h and 48 h in a dose-dependent manner, but lower than that in VL group and SS group at 96 h (P<0.05). CONCLUSION: CSME promotes burn wound healing in the early stages and inhibits the scar hyperplasia in the later stages. The mechanisms may be related to its multicomponents or multiple-targets to intervene in the signaling pathways such as TGF-β₁ regulated by ILK and ILK regulated by PI3K/AKT. It may also be related to the ratio of Col I and Col III expression.     

Effects of angiotensin II type 2 receptor blocker on wound healing in a mouse skin full-thickness injury model

AIM：To observe the effect of angiotensin II (Ang II) and its type 2 receptor (AT2R) on re-epithelialization, granulation tissue formation and growth factor production during wound healing, and to explore the possible mechanism by which Ang II and AT2R influence wound healing. METHODS：Two full-thickness skin wounds were created on the dorsum of C57BL/6J mice. The animals were treated with or without AT2R blocker PD123319 at a dose of 10 mg/kg daily after wounding. Specimens were taken from the wound of each mouse on days 3, 5, 7, 9, 11, 13 and 15 after wounding. Re-epithelialization and granulation tissue formation in the wounded skin tissues were evaluated by HE staining. The production of growth factors,epidermal growth factor (EGF),vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in wounded tissues during the healing process was detected by ELISA. RESULTS：Treatment with PD123319 significantly increased the rate of re-epithelialization and the area of granulation tissue compared with control group at 5 d and 7 d after wounding. Moreover, peritoneal application of PD123319 increased the production of EGF, VEGF and bFGF in the wounded tissues at the indicated time points after wounding. CONCLUSION：AT2R blocker PD123319 accelerates wound healing via promoting re-epithelialization,granulation tissue formation and growth factor production.     

Effect of acupuncture on expression of EGF and bFGF in brain tissues of rats with traumatic brain injury

AIM: To observe the effect of acupuncture on the expression of epidermal growth factor (EGF) and basic fibrolblast growth factor (bFGF) in the brain tissues of rats with traumatic brain injury. METHODS: Thirty SD rats were randomized into sham-operated group, model group and acupuncture group. The model of traumatic brain injury was established by free drop impact. Acupuncture was performed to the rats in acupuncture group once every day and 7 days altogether. Brain histotomy was conducted after the treatment. Immunohistochemical method was adopted to test the protein expression of EGF and bFGF. RESULTS: Compared to sham-operated group, the expression of EGF in the brain tissues of model group decreased (P<0.01), and the expression of bFGF increased (P<0.01). Compared to model group, the expression of EGF and bFGF in acupuncture group increased obviously (P<0.01). CONCLUSION: Acupuncture significantly increases the expression of EGF and bFGF, and improves the repair of injured brain tissues. This might be one of the mechanisms by which acupuncture can treat traumatic brain injury and improve the nervous function.     

Effect of ozone bath on pathological changes and expression of cytokines in rats with deep second-degree burns

AIM: To investigate the effect of ozone bath on the pathological changes and the expression of cytokines, platelet-derived growth factor (PDGF), transforming growth factor-β₃ (TGF-β₃), and tumor necrosis factor-α (TNF-α), in the wounds of deep second-degree burns in rats. METHODS: Male clean-grade SD rats (n=80) were randomly divided into 2 groups, ozone bath group and routine dressing group (control group), with 40 rats in each group. Deep second-degree burn wound was established on the back of the rats, and then the examinations were conducted at 3 d, 7 d, 14 d and 21 d after burn. For the routine dressing group, the wound was cleaned by normal saline and covered with iodophor vaseline gauze every 2 d. For the ozone bath group, before the dressing, the rats were put into the clean foam box to accept ozone fumigation for 20 min (50 mg/L), and then accepted dressing change as the same as that in control group every 2 d. At each time point, the tissue specimens from these rat wounds (at wound center) were taken. The rats in ozone bath group received cleaning by saline cotton and then the ozone bath fumigation, while the rats in control group only received cleaning by saline. After that, the tissue specimens were taken again for HE staining, immunohistochemical staining and semiquantitative observation combined with image data analysis. The concentrations of the cytokines PDGF, TGF-β₃ and TNF-α in the wound were measured by double-antibody sandwich ELISA. RESULTS: In ozone bath group, the wounds were smooth with clear edge and slight inflammatory reaction, swelling and exudation were weaker, and the wound healing rate was higher than that in control group with significant difference. Under microscopic observation with HE staining, slighter inflammatory reaction in ozone bath group was observed than that in control group at each time point, and the numbers of fresh capillaries, fibroblasts and epithelial cells were significantly larger than those in control group. The expression levels of PDGF and TGF-β₃ in the wound tissue homogenate in ozone bath group were higher, and the expression level of TNF-α was significantly lower than those in control group at each time point with significant difference. CONCLUSION: The ozone bath therapy improves the local pathological changes and promotes the expression of cytokines PDGF and TGF-β₃, which are associated with wound healing, as well as reduces the expression of inflammatory mediator TNF-α in the rats with deep second-degree burns, thus promoting the wound healing and anti-inflammatory responses.     

Basic fibroblast growth factor promoting the healing of palatal perforation in rats

AIM: To study the influence of basic fibroblast growth factor(bFGF) on treating ed palatal perforation in rats. METHODS: bFGF was given to the early palatal perforation in rat. The granulation tissues in perforations were grossly and pathologically obserVed. RESULTS: The the of wound healing was significantly in- crease in the bFGF group (P<0.01 ). bFGF could obviously promote the proliferation of the granulation tissues and fibroblasts. The number of AgNORs granules in fibrablasts were 3.73 ±0.52 in the buy group and 2.11 ±0.31 in control group (P < 0.05). CONCLUSION: bFGF could promote the growth of granulation tissues and had a strong promotion on the wound healing in palatal perforation. It was helpful in repairing palatal perforation.     

Effects of basic fibroblast growth factor on proliferation and collagen production in human umbilical cord mesenchymal stem cells

AIM：To observe the growth pattern and surface markers of human umbilical cord mesenchymal stem cells(hUCMSCs) and to explore the influence of basic fibroblast growth factor (bFGF) on the proliferation and collagen production in hUCMSCs cultured in vitro. METHODS：hUCMSCs were isolated by enzyme digestion method and adherent culture. The surface markers CD45, CD34, CD105, CD29 and HLA-DR of the cells were analyzed by flow cytometry. The osteogenic ability and adipogenic differentiation were confirmed with oil red O and alizarin red staining. The optimal concentration of bFGF to promote the proliferation of hUCMSCs was 20 μg/L. The cells in control group were cultured in the growth medium consisting of DMEM/F12 and 10% volume fraction of fetal bovine serum. The cells in experiment group were cultured under the same condition of control group but plus 20 μg/L bFGF. The proliferation of hUCMSCs was analyzed by MTT assay. The expression of type I and III collagens at mRNA and protein levels was determined by RT-PCR and Western blotting. RESULTS：The growth curves indicated that bFGF promoted the proliferation of hUCMSCs. The hUCMSCs expressed CD29, but did not express CD34, CD45 or HLA-DR in the presence or absence of bFGF unanimously. The cells were alizarin red staining-positive and oil red O staining-positive. Compared with control group, the expression of type I and III collagens significantly decreased at mRNA and protein levels in experiment group. CONCLUSION：bFGF promotes the proliferation of hUCMSCs and does not change the expression of the surface markers. bFGF inhibits the expression of type I and III collagens at mRNA and protein levels, indicating that bFGF enhances the healing of wound without inducing scar hyperplasia.     

Activation of α7nAChR promotes wound healing in diabetic mice by suppressing TNF-α expression

AIM: To explore the role of α7 nicotinic acetylcholine receptor (α7nAChR)-specific agonist PNU-282987 in promoting wound healing in diabetic mice by suppressing the expression of tumor necrosis factor α （TNF-α）.METHODS: A model of incised wound was established in diabetic mice or normoglycaemic mice (control). Skin samples were taken on 1 d, 3 d, 5 d, 10 d, 14 d and 21 d post-injury (5 mice in each posttraumatic interval). The numbers of macrophages and fibroblasts, the expression of TNF-α and the deposition of collagen were detected by the methods of immunohistochemistry, Western blotting and Masson staining, respectively. After incised wound was performed in the diabetic mice, PNU-282987 was applied by intraperitoneal injection at suitable posttraumatic interval. The above indexes were investigated again.RESULTS: Compared with control group, the diabetic mice presented delayed wound healing. In diabe-tic mice, the infiltration of macrophages and the expression of TNF-α were significantly reduced in the early phase during wound healing, while they were significantly increased from 5 d post-injury. Besides, from 5 d to 21 d post-injury, the wounds in diabetic mice showed decreased number of fibroblasts and deposition of collagen. From 5 d post-injury, PNU-282987 was applied to diabetic mice, which significantly down-regulated the expression of TNF-α, and increased the number of fibroblasts and the content of collagen in the wounds, eventually promoted wound healing.CONCLUSION: Inflammatory reactions delay wound healing in diabetic mice. Activation of α7nAChR promotes wound healing in diabetic mice by suppressing the expression of TNF-α.     

Expression of VEGF and bFGF in rat model of pressure ulcer

AIM:To study the effects of vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF) on the molecular pathogenesis of pressure ulcer.METHODS:SD rats were randomly divided into control group and experiment group. The pressure ulcer model was established by magnetic disk circulating compression method. HE staining was used to observe the pathological changes of the skin in the rats. The expression of VEGF and bFGF in the tissues was detected by immunohistochemical method. RESULTS:The expression of VEGF and bFGF in the tissues of rat Ⅲ-degree pressure ulcer was lower than that in the surrounding tissues and normal skin(P<0.01). The changes of VEGF and bFGF were consistent(κ=0.58). CONCLUSION:The expression levels of VEGF and bFGF are decreased in the tissues of rat pressure ulcer, suggesting that they may be the potential key factors in the difficult healing of pressure ulcer.     

Effects of bFGF on neuronal apoptosis and fractalkine expression in cerebral ischemia/reperfusion rats

AIM: To study the effects of basic fibroblast growth factor (bFGF) on neuronal apoptosis and fractalkine expression in ischemic penumbra after cerebral ischemia/reperfusion in rats.METHODS: Thirty-six rats were randomly divided into 3 groups: sham operation group, ischemia/reperfusion group and bFGF group. The model of middle cerebral artery occlusion was established by the method of intraluminal filament blockage. The middle cerebral arteries were blocked for 1 h and then reperfused for 24 h. Neurological performances of all rats were scored with Bederson's standard. The brain tissues of the rats were stained and the average infarct volume was calculated. TUNEL method was used to determine the number of apoptotic neurons, and the expression of fractalkine was detected by the method of immunohistochemistry.RESULTS: The score of neurological performances in bFGF group was 2.23±0.59, lower than that in ischemia/reperfusion group (3.18±0.65). The number of apoptotic neurons in bFGF group (13.22±1.35) was lower than that in ischemia/reperfusion group (17.28±1.01, P<0.05), which was the lowest in sham operation group (0.91±0.65). Compared with sham operation group, the expression of fractalkine in ischemia/reperfusion group was decreased. The expression of fractalkine in bFGF group was mainly higher than that in ischemia/reperfusion group (P<0.05).CONCLUSION: Up-regulation of fractalkine may be one of the molecular mechanisms of bFGF to protect neurons against ischemia/reperfusion injury.     

Effect of adriamycin combined with sensitized dendritic cells on cervical tumor-bearing mice

AIM：To explore the immunotherapeutic effect of adriamycin (ADM) combined with frozen-thawed antigen-sensitized dendritic cells (DCs) on cervical tumor-bearing mice. METHODS：The U14 cervical cancer model of Kunming mice was established by subcutaneous implantion of U14 cells in axillary fossa. DCs vaccine was prepared by U14 cervical cancer cell frozen-thawed antigen-sensitized mouse bone marrow-derived DCs. Mature phenotype of sensitized DCs was identified by flow cytometry. Tumor-bearing mice were randomly divided into 4 groups and treated for 3 cycles with PBS (control), DCs vaccine, ADM and ADM combined with DCs vaccine, respectively. The tumor volume was evaluated. The tumor weight and the levels of interleukin-2 (IL-2), IL-12 and interferon γ (IFN-γ) in the serum were determined by ELISA on the 21st day. RESULTS：Cancer cell frozen-thawed antigen-sensitized DCs had higher expression levels of CD11C, CD80 and CD86. The volume and weight of the tumor in ADM combined with DCs vaccine group were less than those in ADM group, DCs vaccine group and control group. The tumor inhibitory rate in combination group was higher than that in the other 3 groups. Compared with the other 3 groups, the serum levels of IL-2, IL-12 and IFN-γ in combination group significantly increased. CONCLUSION：ADM combined with tumor antigen-sensitized DCs vaccine can strengthen the animal antitumor immune response and effectively inhibit the growth of tumor in cervical tumor-bearing mice.     

Expression changes of Wnt/β-catenin signaling pathway in diabetic ulcer

AIM: To observe the effect of Wnt/β-catenin signaling pathway on diabetic ulcer. METHODS: Diabetic animal model was established in the female Wistar rats by intraperitoneal injection of low-dose streptozotocin following high-fat diet feeding. A circular wound was made on the dorsum of the rats in both control group and diabetic group. The condition of wound healing was recorded and the structures of the wound tissues were observed by HE staining in the 2 groups at 3, 7 and 14 d after wounding. The expression of β-catenin, GSK-3β and Rspo-3 at mRNA and protein levels in the wound tissues was detected by RT-PCR and ELISA. RESULTS: In diabetic group, the wound healing rate was lower (P<0.05), and the inflammatory cells, fibroblast cells and new capillaries in the wound tissues were fewer than those in control group. The expression of β-catenin and Rspo-3 at mRNA and protein levels in the wound tissues in control group was significantly higher than those in diabetic group, and the expression of GSK-3β was exactly the opposite (P<0.05). CONCLUSION: The down-regulation of Wnt/β-catenin probably resultes from the decreased level of Rspo-3, which may be one of the reasons for delaying the diabetic ulcer healing.     

Adenosine promotes bFGF protein and bFGF mRNA expression in human umbilical vein endothelial cells in vitro

AIM: To investigate the influence of adenosine on human umbilical vein endothelial cells (HUVEC) bFGF protein production and bFGF mRNA expression. METHODS: Immunohistochemistry staining was performed to detect bFGF protein. RT-PCR was performed to detect bFGF mRNA expression. RESULTS: Immunohistochemistry study demonstrated that there was only a small amount of bFGF positive cells and the color was weak in control group (without adenosine). In groups treated with 10-4 mol/L and 10-6 mol/L adenosine, bFGF protein was significantly higher than that in control group (P<0.05). In 10-8 mol/L and 10-10 mol/L adenosine groups, there were no significant differences compared with control group (P>0.05). RT-PCR showed that in 10-4 mol/L and 10-6 mol/L adenosine groups, bFGF mRNA expression was higher than that in control group (P<0.05), while the difference between 10-8 mol/L adenosine group and control group was not significant (P>0.05). CONCLUSION: Adenosine may promote HUVEC proliferation and angiogenesis partly through inducing bFGF expression.     

Effects of hydrogen sulfide on impaired wound healing in ob/ob mice

AIM: To investigate the role of hydrogen sulfide(H₂S) on impaired wound healing in ob/ob mice and the underlying mechanism.METHODS: The ob/ob mice were randomly divided into 3 groups, including vehicle, insulin and NaHS for treatment. C57BL/6 mice were treated with vehicle as control. Full-thickness punch biopsy wounds were created on the mice. Firstly, H₂S concentrations in the skins and granulation tissues were measured. The mRNA expression of cystathionine γ-lyase(CSE) was detected by RT-qPCR. The protein expression of CSE and MMP-9 were determined by Western blot. The neutrophil and monocyte/macrophage infiltration was analyzed by immunohistochemistry me-thod. The levels of tumor necrosis factor(TNF)-α and interleukin(IL)-6 were measured by ELISA.Collagen formation was measured by Masson staining.RESULTS: The H₂S levels in the skin and granulation were significantly decreased in ob/ob mice and increased in the NaHS-treated mice(P<0.05). CSE expression at mRNA and protein levels was significantly decreased in ob/ob mice compared with the control mice(P<0.05). The wound healing period was significantly shorter in NaHS group than that in vehicle-treated ob/ob mice group(P<0.05), in which the insulin group had no difference with vehicle ob/ob mice group. The neutrophil and monocyte/macrophage infiltration, and TNF-α and IL-6 levels were significantly increased in ob/ob groups, but were decreased in NaHS group(P<0.01 or P<0.05). Meanwhile, NaHS increased collagen formation in the granulation tissues of ob/ob mice.CONCLUSION: H₂S/CSE down-regulation contributes to impaired wound healing in diabetes, which is alleviated by exogenous H₂S possibly through anti-inflammation.     

Endothelial progenitor cell-conditioned medium improves lung structure in neonatal rats exposed to hyperoxia

AIM: To investigate the therapeutic effect of endothelial progenitor cell-conditioned medium (EPC-CM) on the lung structure of neonatal rat exposed to hyperoxia, and to explore the mechanisms.METHODS: Bone marrow-derived endothelial progenitor cells (EPCs) were collected from new born Sprague-Dawley (SD) rats and the EPCs were identified. The conditioned medium from the passage 3 EPCs was collected. Newborn SD rats (n=40) were randomly divided into 4 groups. The rats in room air group were exposed to the room air (21% O₂) for 21 d. The rats in hyperoxia group were exposed to hyperoxia (85% O₂) for 21 d. The rats in endothelial cell basal medium (EBM) group were exposed to hyperoxia for 21 d, and received 100 μL EBM on postnatal day 14 (P14) in a single intratracheal (IT) injection. The rats in EPC-CM group were exposed to hyperoxia for 21 d, and received 100 μL EPC-CM on P14 in a singlie IT injection. The rats were sacrified on the 21st day. The left lungs were excised, placed in 4% paraformaldehyde, serially dehydrated in ethanol and embedded by paraffin. Serial sectioning of the paraffin-embedded left lung tissues was prepared for 5 μm thickness, and stained with hematoxylin and eosin. The pulmonary radical alveolar count (RAC) and alveolar mean linear intercept (MLI) were then calculated. The microvascular density was determined by FVⅢ immunostaining. The mRNA expression of KGF, VEGF, SP-A and SP-C in the right lung tissues was detected by real-time fluorescence quantitative PCR. RESULTS: The cultured cells had typical EPC morphological characteristics, and had the abilities to bind to FITC-UEA-1 and uptake DiI-ac-LDL. The body weight of the rats on day 21, RAC, MLI and microvascular density were significantly lower in hyperoxia group and EBM group than those in room air group (P<0.05). The EPC-CM group had significantly higher RAC and microvascular density than those in hyperoxia group and EBM group (P<0.05), but the body weight and MLI had no significant difference. The mRNA expression levels of KGF, VEGF, SP-A and SP-C in hyperoxia group and EBM group were significantly lower than those in room air group (P<0.05). The mRNA expression levels of KGF, VEGF, SP-A and SP-C in EPC-CM group were significantly higher than those in hyperoxia group and EBM group (P<0.05). CONCLUSION: EPC-CM promotes the lung alveolarization and microvascular formation in neonatal rats exposed to hyperoxia. These benefits may be correlated with the increased KGF and VEGF mRNA expression.     

Effects of alginate dressing and mouse epidermal growth factor combination therapy on epidermal stem cells in patients with refractory wound

AIM: To evaluate the effect of combination therapy with alginate dressing and mouse epidermal growth factor (mEGF) on proliferation and differentiation of epidermal stem cells (ESCs) in patients with refractory wound.METHODS: Eighteen cases,12 males and 6 females,aged from 18 to 61 years (mean 36.4 years),of skin defects therapy by dressing changing for one month were selected in this study.The wounds were 11 in the foot,3 in the calf,2 in the thigh and 2 in the forearm.All the patients were randomly divided into 3 groups: alginate dressing and mEGF group (n=6),mEGF group (n=6) and control group (n=6).The wound closure indexes were measured at 7th,14th,21st and 28th days.The samples were harvested at 7th and 14th days after treatment for pathologic examination.The positive staining cells of cytokeration 10 (CK10) and cytokeration 15 (CK15) were evaluated by SP immunohistochemical staining technique.RESULTS: The wound healing was promoted in group A and group B.However,the wound closure index was increased in group A (P<0.05).Pathological examination showed the epidermis was thicker in group A than that in group B and C.Treatment by the alginate dressing combined with the mEGF,angiogenesis was active and granulation was enhanced.As compared with group B and group C,SP immuohistochemical staining technique showed that the ESCs in group A were bigger in size and larger in number.There was significant difference between group A and group B (or C) in the proliferation and differentiation of ESCs.CONCLUSION: Combined therapy with alginate dressing and mEGF shows a better result than that with mEGF only in proliferation and differentiation of ESCs of patients with refractory wound.     

Expression of calpain 2 and Bax in rat fibrotic liver tissues

AIM：To observe the expression of calpain 2 and Bcl-2-associated X protein (Bax) in rat fibrotic liver tissues and to explore their effects on hepatic fibrosis.
METHODS：Forty male Wistar rats were randomly divided into four groups (each n=10): 4-week control group, 8-week control group, 4-week liver fibrosis group and 8-week liver fibrosis group. Liver fibrosis model was induced by subcutaneous injection of 40% CCl4 (3 mL/kg) every 3 days for 4 or 8 weeks. The apoptosis of hepatocytes was detected by TUNEL. Additionally, the mRNA expression of calpain 2 and bax was determined by real-time PCR, and the protein expression of calpain 2 and Bax was determined by immunohistochemistry and Western blotting. RESULTS：Real-time PCR showed that the mRNA expression of calpain 2 and bax in liver tissues was elevated in 4-week and 8-week liver fibrosis groups. The results of immunohistochemistry and Western blotting revealed that there was no difference of calpain 2 protein expression in liver tissues between 4-week liver fibrosis group and control group, but that in 8-week liver fibrosis group was obviously increased. The protein expression of Bax in 4-week and 8-week liver fibrosis groups was higher than that in control groups. Additionally, the numbers of apoptotic hepatocytes in 4-week and 8-week liver fibrosis groups were obviously increased compared with control groups.CONCLUSION：Calpain 2 and Bax may play important roles in the process of liver fibrosis.     

Effect of ligustrazine on expression of LFA-1 and ICAM-1 in bone marrow of radiation injured mice

AIM: To investigate the effect of ligustrazine on the expression of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) in bone marrow and on the mechanism of hematopoietic reconstitution in radiation injured mice.METHODS: The 24 mice (clean class) were randomly divided into 3 groups: normal group, radiation injured group and ligustrazine group. After irradiation by 6.0Gy ［⁶⁰Co］ γ-ray, the radiation injured animals were given normal saline (0.2 mL, twice a day) through gastric tube, while the ligustrazine group was given ligustrazine through gastric tube (0.2 mL, twice a day). The mice in normal group received no treatment. At the 7th, 14th, 21st day after irradiation, the femur were taken and the bone marrow mononuclear cells (BMNCs) suspension were made to culture bone marrow stromal cells (BMSCs). The mRNA and protein expressions of ICAM in BMSCs were assayed by RT-PCR and Western blotting. The expression levels of LFA-1 in BMNCs were evaluated by flow cytometry analysis.RESULTS: In ligustrazine group the expression levels of LFA-1 at the 7th, 14th and 21st days after irradiation were higher than those in radiation injured group (P<0.01 or P<0.05). However, the expression level of ICAM-1 was lower than that in the compared group (P<0.01 or P<0.05).CONCLUSION: Ligustrazine can increase the LFA-1 expression level of BMNCs, decrease the ICAM-1 expression level in BMSCs, indicating that ligustrizine promotes the recovery of hematopoietic cells in bone marrow, then improves the bone marrow microenvironment and enhances hematopoietic reconstitution.     

Effects of bFGF on brain edema,nerve function injury and autophagy related protein in rats with traumatic brain injury

AIM:To study the effects of basic fibroblast growth factor (bFGF) on brain edema, nerve function damage and autophagy related proteins in rats with head injury. METHODS:The rat model of craniocerebral injury (CI) was constructed. The rats were divided into control group, CI group, and low-, middle-and high-dose bFGF groups (n=10). The CI model was established in CI group, while the rats in control group were not given epidural impact. The rats in low-dose, middle-dose and high-dose bFGF groups were given bFGF at 2, 4 and 6 μg, respectively, by intraperitoneal injection after 30 min. The neurological function in the rats was evaluated by improved neurological function scoring. The rat brain tissues were taken, and the water content was detected. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in the brain tissue were measured by ELISA. The malondialdehyde (MDA) content was analyzed by thiobarbituric acid method. The activity of superoxide dismutase (SOD) was examined by WST-8 assay. The glutathine peroxidase (GSH-Px) activity was detected by colorimetric method. The protein levels of autophagy related proteins LC3-Ⅱ and beclin-1 in the brain tissues were determined by Western blot. RESULTS:The neurological function score was increased significantly of the rats in CI group. The rat model of craniocerebral injury was successfully constructed. Neurological function scores in the rats in low-dose, middle-dose and high-dose bFGF groups were reduced, the water content of the brain tissue was also reduced (P<0.05). The levels of TNF-α, IL-6 and IL-1 β were decreased in the brain tissues (P<0.05), the content of MDA was declined (P<0.05), the activities of SOD and GSH-Px were increased (P<0.05), the protein levels of LC3-Ⅱ and beclin-1 were decreased, compared with the untreated rats in CI group (P<0.05). CONCLUSION:bFGF improves the nerve function of the rats with craniocerebral injury, reduces the water content of the brain tissue, reduces the expression of autophagic protein LC3-Ⅱ and beclin-1.The mechanism is related to the inhibition of inflammatory reaction and oxidative damage.     

Ectopic hTERT gene expression in human bone marrow mesenchymal stem cells

AIM: To investigate the effect of transfection of hTERT gene into human mesenchymal stem cells (MSCs) on their telomerase activity and life-span.METHODS: Human MSCs were transfected with a pEGFP-hTERT plasmid by liposome-mediated transfection. Then the hTERT mRNA expression in MSCs was detected by RT-PCR. The activity of telomerase in transfected MSCs was detected by PCR and ELISA. The telomerase-positive MSCs was cultured in vitro and induced into neuron-like cells with EGF and bFGF. Neuron-specific markers (NF-M, MAP₂) were detected by RT-PCR.RESULTS: hTERT fragment was identified in the hTERT-transfeced cells but not in the untransfected human bone marrow MSCs. The untransfected human MSCs remained telomerase-negative but the hTERT-transfected cells showed robust telomerase activity. The telomerase-negative MSCs entered a nondividing state and senesced after about 20 to 25 passages. In test group, however, telomerase-positive MSCs to date had undergone 35 passages. RT-PCR analysis showed that telomerase-positive MSCs expressed neuron-specific markers, such as NF-M or MAP₂ after induced with EGF and bFGF in vitro. CONCLUSION: Ectopic expression of the hTERT gene in human MSCs reconstitutes telomerase activity. The transfection of hTERT gene into human MSCs extends their replicative life span and maintains their multipotent differentiation capacity.