Effect of different doses of adipose-derived stem cells on early renal fibrosis / 中国组织工程研究

BACKGROUND:Our preliminary findings have shown that stem cells have a certain effect on early formation of renal fibrosis and delay the occurrence of renal interstitial fibrosis.OBJECTIVE:To investigate the effect of exogenous adipose-derived stem cell transplantation on the formation of renal interstitial fibrosis in rats,and to explore the dose-effect relationship.METHODS:Fifty Sprague-Dawley rats were randomly divided into five groups.Rats in sham operation group were injected with PBS;and those in the other four groups were ligated to establish the model of renal fibrosis.After successful modeling,the model group was injected with PBS,while low dose group,middle dose group and high dose group were injected with 1 ×107/L,2x107/L,3x107/L adipose-derived stem cell suspension (0.1 mL),respectively.Rats were killed 14 days after injection.Hematoxylin-eosin staining and Masson staining were used to observe the degree of renal tubular interstitial injury and the relative area of the renal interstitium.Expression of alpha smooth muscle actin and transforming growth factor beta1 was detected by immunohistochemistry.RESULTS AND CONCLUSION:(1) Hematoxylin eosin staining:The model group showed typical glomerular and tubulointerstitial changes;the low,middle and high dose groups had a certain degree of glomerular and tubulointerstitial changes,but the severity of injury in these three groups,especially in the high dose group,was significantly milder than that in the model group.(2) Masson staining:In the model group,the renal interstitium was widened and the collagen fibers were deposited;in the low,middle and high dose groups,the degree of renal interstitial expansion was lower than that in the model group,and moreover,the degree of renal interstitial expansion in the middle and high dose groups was lower than that in the low dose group (P ＜ 0.05).(3) Immunohistochemical staining:Compared with the sham operation group,the expression of alpha smooth muscle actin and transforming growth factor beta1 in the model group was higher than that in the sham operation group (P ＜ 0.05).Compared with the model group,significantly decreased expression of alphasmooth muscle actin was found in the high dose group (P ＜ 0.05) and significantly reduced transforming growth factor beta1 expression was observed in the high,middle,and low dose groups (P ＜ 0.05),especially in the middle and high dose groups.To conclude,exogenous adipose-derived stem cell transplantation can delay the formation of early renal fibrosis in a dose-effect manner,which may be realized by changing the signal pathway state induced by transforming growth factor beta1.

Effect of integrin β8 on TGF-β1 activation in astrocytes with oxygen glucose deprivation / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effect of β8 expression on transforming growth factor β1(TGF-β1) activation in astrocytes with oxygen glucose deprivation (OGD).</p><p><b>METHODS</b>Astrocytes were cultured and then subjected to OGD to generate hypoxia-ischemia (HI) model in vitro. Immunocytochemistry was used to detect the expression and distribution of β8 in nomoxia cultured cells. β8 protein expression was quantified by Western blot at 12 hours, 1 day and 2 days after OGD. Astrocytes and luciferase reporter cells (TMLC) were co-cultured. β8 RNA interference system was established to specifically inhibit β8 expression in cultured astrocytes. TGF-β1 activation was then detected in the co-culture system.</p><p><b>RESULTS</b>β8 was mainly located in the cytoplasm and neurites of astrocytes. OGD resulted in increase of β8 protein expression at 12 hours after reoxygenation in astrocytes, which was peaked at 1 day after reoxygenation. TGF-β1 activation was in accordance with β8 expression in astrocyte-TMLC co-culture system after reoxygenation. After the inhibition of β8, TGF-β1 activation was significantly reduced in all time points.</p><p><b>CONCLUSIONS</b>The highly expressed β8 plays important roles in the regulation of TGF-β1 activation in neonatal rats with hypoxic-ischemic brain damage.</p>

Role of the FOXO3a transcription factor in neuronal apoptosis in neonatal rats with hypoxic-ischemic brain damage / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore the role and mechanisms of FOXO3a nuclear translocation in neuronal apoptosis after hypoxia-ischemia (HI).</p><p><b>METHODS</b>One hundred and sixty 10-day-old Sprague-Dawly rats were randomly divided into two groups: HI and sham-operated. The right common carotid artery was ligated followed by hypoxia exposure for 2.5 hours in the HI group. The sham-operated group rats were not subjected to carotid artery ligation or hypoxia treatment. Rat cerebral cortex was collected at 0.5, 2, 4, 8 and 24 hours after hypoxia. Western blot was used to detect expression of total FOXO3a protein, pnuclear and cytoplasmic FOXO3a and Bim. TUNEL staining was used to detect apoptotic cells.</p><p><b>RESULTS</b>The nuclear protein of FOXO3a obviously increased from 0.5 to 24 hours after HI in a time-dependent manner compared with the sham-operated group (P<0.01). On the contrary, cytoplasmic protein evidently decreased from 0.5 to 24 hours in the HI group compared with the sham-operated group (P<0.01). Bim protein increased from 0.5 hour, peaked at 2 hours, started to decline at 4 hours (P<0.01), and returned to baseline level at 8 and 24 hours after HI in the HI group compared with the sham-operated group. TUNEL positive cells started to express at 4 hours, and peaked at 24 hours after HI (P<0.01). However, TUNEL positive cells were rarely found in the sham-operated group.</p><p><b>CONCLUSIONS</b>HI induces FOXO3a translocation from cytoplasm to nucleus, and enhances protein expression of its target gene Bim in the neonatal rat brain. The upregulation of Bim expression might be related to neuronal apoptosis.</p>

Clinical application of inhaled nitric oxide in hypoxic respiratory failure of preterm infants / 中国当代儿科杂志

Inhaled NO (iNO) has been shown to have beneficial effects on decreasing pulmonary inflammation, increasing function of surfactant and improving lung growth in prematurely born animal models. iNO has been gradually applied in the neonatal intensive care unit since its first use for persistent pulmonary hypertension (PPHN) in the early 1990's. Although many research findings have shown the benefits of iNO for hypoxic respiratory failure (HRF) of preterm infants, there is no certain evidence to support the routine use of iNO in premature infants. According to recent literature, the mechanism of iNO therapy, treatment scheme, iNO effectiveness and safety in premature infants were reviewed in this article, so as to provide bases for the clinical use of this treatment.

Study on transport of Yangyin Tongnao granules in main effective fractions / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the absorption characteristics and transportation mechanism Yangyin Tongnao granules in main effective fractions in Caco-2 cell model.</p><p><b>METHOD</b>The safety concentrations of Yangyin Tongnao granules in main effective fractions in Caco-2 cells. A Caco-2 cell model was established to study the transport situations after the compatibility of Yangyin Tongnao granules in main effective fractions, and the content was determined by high performance liquid chromatography (HPLC).</p><p><b>RESULT</b>P(app) of puerarin, ligustrazine and astragaloside were less than 1.0 x 10(-6) cm x s(-1), and their P(app) were hard to be close to atenolol. The oral absorption in descending order is shown as the following: puerarin, ligustrazine, astragaloside. After the compatibility between saponins and flavonoids, P(app) of astragaloside was improved obviously, which promoted the transport from apical (AP) to basolateral (BL); the compatibility of puerarin, ligustrazine and astragaloside showed a significant effect in the efflux of astragaloside and no change in the absorption transport of ligustrazine and puerarin at the same time. There is a great difference in bidirectional transport of representative component of each effective fraction, and P(app)(B --> A) was significantly greater than Papp(A --> B), which suggested that the efflux transport from BL side to AP side had an advantage in the three representative components of the three effective fractions in Caco-2 cell monolayer model.</p><p><b>CONCLUSION</b>Astragaloside, ligustrazine and puerarin may be malabsorptive compounds, and the three compounds may be discharged by the transport protein in small intestine membrane.</p>

Chuanxiongzine-astragaloside IV decreases IL-1β and Caspase-3 gene expressions in rat brain damaged by cerebral ischemia/reperfusion: a study of real-time quantitative PCR assay / 生理学报

The purpose of this study was to establish an absolute quantitative method to detect IL-1β and Caspase-3 gene expressions in rat brain after cerebral ischemia-reperfusion (I/R) using real-time PCR. Rats were randomized into the following groups: sham operation group, model group (cerebral I/R group), astragaloside IV (AST IV) group, chuanxiongzine-AST IV group and nimodipine group (n = 10 in each group). The rats in all the groups except sham operation group were subjected to cerebral I/R treatment. Sham operation and model groups were treated by normal saline (5 mL/kg). AST IV, chuanxiongzine-AST IV and nimodipine groups received 20 mg/kg AST IV, 10 mg/kg chuanxiongzine plus 20 mg/kg AST IV, and 10 mg/kg nimodipine treatments, respectively. The administrations of drugs were performed with intraperitoneal injections at 0 and 12 h, 1 d, 2 d, 3 d, till to 7 d after I/R. A real-time quantitative PCR assay was developed for absolute quantification of the expressions of IL-1β and Caspase-3 genes. The absolute quantification approach relies on the construction of an accurate standard curve. Thus, two plasmids which contained rat IL-1β and Caspase-3 genes respectively were constructed. The cloned circular plasmids were then quantified using a spectrophotometer and used as standards. Standard curves were generated, and the copy numbers of IL-1β and Caspase-3 mRNA isolated from I/R-damaged brain tissue were also calculated by SYBR Green I dye method using specific primers. The results showed that melting curves exhibited sharp peaks, and PCR product also generated prominent band with expected size in agarose gel electrophoresis, which validated the optimization of the selected primer sets of IL-1β and Caspase-3 genes. The optimal annealing temperatures of IL-1β and Caspase-3 genes were 59 °C and 61.2 °C, respectively. Real-time PCR results showed that the expression of IL-1β and Caspase-3 genes in the model group was significantly elevated compared to that in the sham operation group. However, compared to those in the model group, IL-1β and Caspase-3 gene expressions were obviously decreased in AST IV, chuanxiongzine-AST IV and nimodipine groups. Especially in chuanxiongzine-AST IV group, those two genes showed the most significant expression down-regulation. These results suggest the absolute quantitative method established in the present study is capable of detecting the changes of IL-1β and Caspase-3 gene expressions in rat brain damaged by I/R.

Roles of glutamate receptor 2 and cellular free calcium in the pathogenesis of periventricular leukomalacia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the expression of the AMPA receptor subunit glutamate receptor 2 (GluR2) and the cellular free calcium concentration in the white matter of neonatal rats with periventricular leukomalacia (PVL) and their roles in the pathogenesis of PVL.</p><p><b>METHODS</b>A PVL model was prepared by unilateral carotid artery ligation (UCL) followed by exposure to 6% oxygen for 4 hrs in 2-day-old rats. The neonatal rats performed a sham operation, without hypoxia-ischemia (HI), were used as the control group. At 12, 24, 48 and 72 hrs of HI, the expressions of GluR2 mRNA and protein in the white matter were detected using real time quantitative PCR and Western blot respectively. Spectrophotofluorimetry and Fura 2/AM were used to detect the cellular free calcium concentration.</p><p><b>RESULTS</b>The expressions of GluR2 mRNA and protein in the white matter were significantly reduced in the PVL group at 24 hrs of HI, and remained at lower expressions until 72 hrs of HI compared with the control group (P < 0.05). The cellular free calcium concentrations increased significantly in the PVL group at 12 hrs of HI, and remained at higher levels until 72 hrs of HI compared with the control group (P < 0.05).</p><p><b>CONCLUSIONS</b>The expressions of GluR2 mRNA and protein in the white matter decreased whereas the cellular free calcium concentration increased in neonatal rats with PVL. The decreased expression of GluR2 might lead to the overloading of cellular calcium in the white matter, which may cause neuronal damage and death.</p>

Expression of P75NTR protein and RhoA mRNA in the brain of neonatal rats with white matter damage / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Recent studies have indicated that the signal pathway of NgR-P75NTR- RhoA plays a key role in nerve injury and remodeling, but its exact mechanism and the role of the downstream molecule RhoA regulated by P75NTR remain unclear in hypoxia-ischemia (HI) neonatal animals. The present study was designed to assess the expression of P75NTR protein and RhoA mRNA in neonatal white matter and to investigate their relationship in newborn rats with white matter damage (WMD).</p><p><b>METHODS</b>The rat WMD model was established by the ligation of right common carotid artery, followed by 6% hypoxia exposure for 4 hrs. The control group was sham-operated, without HI treatment. The histological changes of brain tissue were observed under light and electron microscopes. Expression of P75NTR protein and RhoA mRNA in the brain white matter after 12, 24, 48 and 72 hrs and 7 days of HI were detected by RT-PCR and immunohistochemistry, respectively.</p><p><b>RESULTS</b>Periventricular white matter damage was observed by 48 hrs of HI. Expression of P75NTR protein increased in the striatum and callosum zones at 12 hrs, peaked at 48 hrs, and remained at a higher level than control until 72 hrs of HI in the WMD group (P < 0.01). After 7 days of HI expression of P75NTR protein was no longer statistically different from controls. The RhoA mRNA was higher in the WMD group for the first 72 hrs and then declined to control values.</p><p><b>CONCLUSIONS</b>Increased P75NTR protein might mediate apoptosis of nerve cells and inhibit the regeneration of neuron axons. The subsequent decline back to control value may be correlated with the aggregation of necrosis of nerve cells after HI. The patterns of RhoA mRNA expression were consistent with those of P75NTR protein, suggesting that the increased P75NTR level may promote RhoA mRNA expression.</p>

Expression of F-actin and RhoA in experimental hypoxic-ischemic white matter damage in premature SD rats / 中华儿科杂志

<p><b>OBJECTIVE</b>White matter damage (WMD) in preterm infants is a well-recognized serious complication of prematurity. The collapse of cell skeleton of growth cone after hypoxia-ischemia (HI) is considered as the basic neuropathologic change of the long-term residuals of premature white matter damage. F-actin is the major component of cell skeleton and maintains the normal form of cells, its function and potential mechanism of WMD have not been reported. In this study, changes of F-actin and its influencing factor RhoA were investigated.</p><p><b>METHODS</b>Totally 184 Sprague-Dawley (SD) rats (age 2 days, body weight 6 to 8 grams) were randomly divided into 14 groups: 7 different time WMD groups (HI 12 h, 24 h, 48 h, 72 h, 7 d, 14 d, 28 d) and 7 corresponding control groups. The 2 day-old SD rats were subjected to ligation of right carotid artery (ischemia), and then they were put into a box full with 6% oxygen and 94% nitrogen for 4 hours (hypoxia). The light microscopy was used to observe the brain pathological changes and the electron microscopy was used to detect the brain ultrastructural changes after hypoxia and ischemia. Eighty SD rats were used for flurescent-immunohistochemical method to detect the distribution of F-actin in cell membrane and cytoplasm of both WMD groups and the control groups at 12 h, 24 h, 48 h, 72 h, 7 d after HI respectively. The distribution of F-actin was reflected by the percentage of non-integrity cells. Another 80 SD rats were used for real time RT-PCR to detect the expression of RhoAmRNA in the white matter tissue of both WMD groups (HI 12 h, 24 h, 48 h, 72 h, 7 d) and the control groups.</p><p><b>RESULTS</b>(1) Necrosis of lateral ventricle tissue was observed by 72 h after HI. Dilatation of ventricle and formation of capsular space beneath white matter had been observed by 14 d after HI. (2) Disregulation, pyknosis, mitochondrion swelling and chromatin agglutination were observed in WMD groups. The maldevelopment of myelins in WMD groups was detected at 1 h after HI. (3) The fluorescent stains decreased on cellular membrane, but increased in cytoplasm with time. The percentage of non-integrity cells was significantly higher (P < 0.05) in HI groups (0.32 +/- 0.04, 0.43 +/- 0.04, 0.56 +/- 0.03, 0.65 +/- 0.04, 0.87 +/- 0.03) than the controls (0.02 +/- 0.01, 0.02 +/- 0.01, 0.01 +/- 0.01, 0.02 +/- 0.01, 0.02 +/- 0.01). (4) The expression of RhoA mRNA was significantly increased (P < 0.05) in HI groups (1.205, 2.415, 4.830, 1.500) in the white matter tissue compared with the controls (0.300, 0.375, 0.375, 0.530) at 12 h, 24 h, 48 h, 72 h after HI. The expression of RhoA mRNA reached the peak value at HI 48 h, and then gradually decreased. The expression of RhoA mRNA at HI 7 d in WMD group (0.500) was not significantly different from the control (P > 0.05).</p><p><b>CONCLUSION</b>(1) The pathological and ultrastructural changes of white matter in WMD groups after HI suggest that the WMD model was successfully set up in premature 2 days SD rats. (2) F-actin is redistributed within cells after HI: expression in membrane is decreased and expression in plasma was increased. The redistribution possibly results in the collapse and retraction of cells. (3) The expression of RhoA mRNA is increased significantly after HI, which may lead to the redistribution of F-actin. (4) The increase of the expression of RhoA mRNA is not persistent, but the redistribution of F-actin is continued, which suggests that RhoA may not be the only factor affecting the redistribution of F-actin.</p>

Establishment and Evaluation on Experimental Newborn Rats Model of Periventricular Leukomalacia / 实用儿科临床杂志

Objective To establish the 2 day-old SD rats model of periventricular leukomalacia(PVL).Methods Sixty-five healthy 2 day-old SD rats were randomly divided into 3 experimental PVL groups and 3 control groups.The experimental PVL groups were subjected to right carotid ligation(RCL),and then they were suffered from hypoxia by 6% oxygen and 94% nitrogen for 4 hours.Meanwhile sham surgeries were performed on control groups without exposed to hypoxia.Light and electronic microscopy were used to observe brain pathological changes.Immunohistochemistry methods were used to detect the distribution and expression of glial fibrillary acidic protein(GFAP),?-amyloid precursor protein(?-APP),myelin basie protein(MBP) and O4 of brain at 72 hours post-operation.Han-(ging) test,inclined plane test,open field test and cylinder test were performed on the rats 28 days post-operation.Results 1.In the PVL groups,light and electronic microscopy showed that tissue necrosis was observed in the periventricular white matter area at early stage,and at later stage right ventricular dilation,decrease of the corpus callosum area and loss of medullary sheath were detected.The morphometrical analysis showed that GFAP and ?-APP integrated optical density(A) of PVL group was increased,and mean diameter of GFAP-immunoreactive cells was also increased in PVL group,while MBP A of PVL group was decreased compared with contral group.The density of O4-immunoreactive abnormal cells was dramatically increased in PVL group.The outcomes of neurobehavioral tests of PVL group were greatly abnormal compared with control group.Conclusion The changes in 2-day-old SD rats,RCL-hypoxia model are accor-(ded) with the pathology and behavior characeriatis of PVL.

Relative Factors of Intraventricular Hemorrhage in Premature Infants / 实用儿科临床杂志

Objective To analyze the etiologic factors and clinical manifestation of intraventricular hemorrhage (IVH) in preterm infants.Methods One hundred and seventy-two preterm infants from June 2005 to August 2006 were accrued to investigate their gestational age,birth weight,birth history,and clinical symptoms.Cranial chromatic ultrasound was used to scan the preterm infants and diagnose IVH.Results 1.The incidence of IVH was associated with gestational age (?2=6.40 P=0.011);2.The incidence of IVH was also associated with birth weight(?2=26.49 P=0);3.IVH usually occurred within 72 h with mild clinical manifestations and was diagnosed within 5 days after birth;4.IVH occurred more frequently and more severe in infants with severe asphyxia than those with mild asphyxia.Conclusions Early gestational age,low birth weight, and severe asphyxia are risk factors for IVH.The clinical symptoms of IVH are usually mild in most patients.Cranial chromatic ultrasound is a reliable,sensitive and convenient method of detection for IVH in preterm infants.