Volume 93  ·   Issue 20  ·  2013

To explore the inhibitory effects of tacrolimus (FK506) on effector T cells in vitro and examine the relationship between effector T cells and clinical features in patients with severe aplastic anemia (SAA) to elucidate its immune mechanism.

The CD8⁺HLA-DR⁺cells, sorted by immunomagnetic separation from bone marrow mononuclear cells (BMMNC) of 16 SAA patients, were cultured in different concentrations of interleukin-2 (IL-2) alone or with FK506 for 72 hours.The proliferation effect was measured with methyl thiazolyl tetrazolium (MTT) method.The T lymphocytes were sorted from the SAA patients by lymphocyte separation medium and cultured alone or with IL-2 or with FK506 or FK506 plus cyclosporin A (CsA) for 18 hours.The expression of tumor necrosis factor-β (TNF-β) in CD8⁺HLA-DR⁺T cells was analyzed by flow cytometry.The relationship between the expression of TNF-βand the clinical data, including percentages of reticulocyte and lymphocytes in peripheral blood cell count and ratio of CD4⁺T cells and CD8⁺T cells, was also analyzed.

At the concentration of IL-2 greater than or equal to 20 U/ml, the cell proliferation (A values,0.538±0.142) were significantly higher than that in the blank culture hole (0.505±0.153)(P<0.05).The A values significantly decreased (0.386±0.124) after the addition of FK506 (P<0.05).Compared with control group, the expression of TNF-β was significantly higher in IL-2 group (73.36%±16.73% vs 66.61%±16.20%,P<0.05), significantly lower in FK506 and FK506 plus CsA groups (P<0.05).No significant differences existed between the FK506 and FK506 plus CsA groups (47.78%±20.09% and 42.23%±21.35%, P>0.05).The expression of TNF-β in SAA was negatively correlated with the percentage of reticulocyte and the ratio of CD4⁺T cell and CD8⁺T cell, positively correlated with the percentage of lymphocyte in peripheral blood count (r=-0.86,-0.90,0.77,all P<0.05).

IL-2 can enhance the proliferation and expression of TNF-β of CD8⁺HLA-DR⁺T cells from SAA patients.Such an effect is inhibited by FK506.And FK506 and FK506 plus CsA have similar effects.

To explore the proportion of Th22 cells in peripheral blood of patients with aplastic anemia (A A) and evaluate its significance.

From January 2011 to June 2012,a total of 47 AA patients were recruited and divided into 4 groups:severe aplastic anemia (SAA) pretherapy (group A,n=11),non-severe aplastic anemia (NSA A) pre-therapy (group B,n=12),SAA post-therapy (group C,n=12), NSAA post-therapy (group D,n=12) and healthy donor controls (n=12).The proportion of Th22 cells in peripheral blood of each group was evaluated by flow cytometry.The cytokines interleukin-22 (IL-22), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured by ELISA.And the level of IL-22 mRNA was examined by reverse transcription-PCR (RT-PCR).

The percentage of Th22 cells and the level of IL-22, TNF-α, IL-6 and IL-22 mRNA in group A (4.3%±1.4%, (57±17) ng/ L, (497±123) ng/L, (323±88) ng/L, 1.65±0.51) and group C (2.6%±0.6%, (34±10) ng/L, (314±79) ng/L, (187±45) ng/L, 0.92±0.28) were significantly higher than that in control group (1.2%±0.3%, (19±6) ng/L, (228±50) ng/L, (134±26) ng/L, 0.47±0.09,all P<0.05).The percentage of Th22 cells and the level of IL-22, TNF-α, IL-6 and IL-22 mRNA in group A were higher than those in group C (all P<0.05).NSAA patients had similar results.The percentage of Th22 cells and the level of IL-22, TNF-α, IL-6 and IL-22 mRNA in group A were higher than those in group B (all P<0.05).But the level of TGF-βin groups A and C were significantly lower than that in control group ((3.4±1.1) and (5.8±1.7) vs (9.7±2.8) ng/L,P<0.05).And the level of TGF-βin group A was lower than that of group B (P<0.05).

The number of Th22 cells is elevated in AA patients.Th22 cells may be positively correlated with the development of AA.And a higher level of TNF-α, IL-6 and a lower level of TGF-βpromote the differentiation of Th22 cells.

To explore the changes in telomere length and gene expression of complex shelterin (composed of 6 core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1) in severe aplastic anemia (SAA).

Bone marrow samples were obtained from 20 SAA patients and 10 normal controls.CD3⁺T cells were sorted by immunomagnetic separation.Telomere length was tested by Southern blot and the gene expressions of TRF1, TRF2, POT1, TIN2, TPP1 and RAP1 were detected by reverse transcription-PCR (RT-PCR).

Telomeres of CD3⁺T cells were found significantly shorter in SAA untreated ((4.4±1.1) kb,n=9) and recovering groups ((5.8±1.0) kb,n=11) than control group ((9.2±3.3) kb, P<0.05).Telomere length of CD3⁺T cells shortened with T_H/S decreasing (r=0.564, P=0.029).The mRNA expression of POT1 decreased in untreated SAA patients (0.16 (0.02-0.29)) and over-expressed in recovering patients (1.17 (0.82-1.86),P<0.05).The mRNA expression of RAP1 was significantly higher in untreated patients (4.14 (1.93-6.92)) than that in recovering group (0.87 (0.30-1.73) ) and controls (0.62 (0.45-4.07), both P<0.05).

Changes in telomere length and shelterin gene expression occur in CD3⁺T cells of SAA patients and may be correlated with disease severity.

To evaluate the possible mechanism of transcription factors B cell lymphoma 6 (Bcl-6), forkhead/winged helix transcription factor 3 (Foxp3) and retinoic acid related orphan receptor (RORγt) in CD4⁺T cells for immuno-related hematocytopenia (IRH).

CD4⁺T cells were harvested from 40 IRH patients, 38 aplastic anemia subjects and 25 normal controls and separated by magnetic activated cell sorting (MACS).Then the expressions of transcription factors of Foxp3, RORγ and Bcl-6 in CD4⁺T cells were measured by real time fluorescent quantitative-polymerase chain reaction (QRTPCR).

Auto-antibody was detected on CD34⁺cells (67.5% (27/40)), CD15+ cells (65.0% (26/40)), Gly A⁺cells (75.0% (30/40)),auto-antibody involving three, two or one myeloid cell were detected in 27.5% (11/40),52.5% (21/40),20.0% (8/40) of IRH patients.Compensatory increase of Foxp3 mRNA was found in IRH (0.124 (0.073-0.198) vs 0.071 (0.046-0.118),P<0.05).The expression of Bcl-6 was higher (2.243 (0.854-4.544) vs 1.211 (0.131-2.816),P<0.05).Compared to aplastic anemia, the expression of RORγt was lower in IRH (0.133 (0.068-0.189) vs 0.290 (0.138-0.480),P<0.01) and the ratio of Treg/Th17 shifted to Th17 in patients with aplastic anemia (Foxp3/RORγt ratio,0.500 (0.240-0.795) vs 0.975 (0.483-1.416),P<0.01).

As one kind of bone marrow failures caused by autoantibody to bone marrow cells, IRH may occur due to a high expression of Bcl-6 in CD4⁺T cells,its immunopathogenesis is different from that of aplastic anemia.

To evaluate the feasibility and clinical results of subsequent retroperitoneoscopic surgery for patients with previousipsilateral retropenitoneal surgery through frank incision.

A total of 10 patents were selected for subsequent laparoscopic surgery through retroperitoneal approach.Among them, there were recurrent renal cysts (n=4) including a history of open surgery (n=1) and retropenitoneal surgery (n=3) and nonfunctional kidneys (n=6) including open nephropyelopolasty (n=3), retroperitoneoscopic nephropyelopolasty (n=1) and retroperitoneoscopic ureterolithotomy (n=2).The mean surgical duration was (12-85) 38.6 months.All patients underwent retroperitoneoscopy.Decortication was performed for renal cysts and nephrectomy for nonfunctional kidneys.

All operations were successfully performed with a mean surgical duration of 97 (40-185) minutes and a mean volume of blood loss 125 (20-460) ml.Among 4 cases with intraoperative peritoneal rupture, one case had renal cyst on ventral side.After enlargement, the procedure was performed through peritoneal cavity.The mean postoperative hospital stay was 5.6 (3-9) days.Nine patients received a mean follow-up period of 21.5 (3-47) months.All symptoms were relieved without any occurrence of postoperative complications.

For patients with previous ipsilateral retropenitoneal surgery, retroperitoneoscopy may be feasible for properly selected cases.

To explore the causes and treatment options of acute renal failure induced by unilateral ureteral calculi obstruction.

The clinical data of 12 cases of acute renal failure induced by unilateral ureteral calculi obstruction between August 2008 and July 2012 were reviewed retrospectively.There were 5 males and 7 females with an average age of 65.7 years.Their clinical data and treatment options were retrospectively analyzed and summarized.Seven cases showed right side ureteral calculus with hydronephrosis while another 5 presented left side ureteral calculus with hydronephrosis.Serum creatinine was higher than 310μmol/L in 12 cases.Anuria appeared in 4 cases for 1-7 days while oliguria in 8 cases for 2-10 days.High fever was present in 11 cases, the highest of whom was 40℃.White blood cell count increased in 10 cases (>10×10⁹/L) and decreased in 2 cases (<4×10⁹/L).

The therapeutic options included insertion of double J stent for internal drainage (n=1), percutaneous nephrostomy for external drainage (n=10) and open operation (n=1).Traditional treatments were performed to manage ureteral calculus in the above 11 cases with drainage.All cases had improved renal function after comprehensive treatment of anti-infection, antishock, rinsing stones and relieving obstruction.All 12 cases were treated successfully.

Unilateral ureteral calculus may impair contralateral renal function and cause acute renal failure due to the absorption of toxin at obstructive side.The keys of management are eliminating toxin and relieving obstruction.

To evaluate clinical outcomes and values of transperitoneal laparoscopic ureterolithtomy.

A total of 1171 patients with upper ureteral calculi were recruited during September 1999 to February 2012.The etiologies included impacted calculi (n=1100), non-impacted calculi (n=71), multiple ureteral calculi (n=51) and combined small calculi in unilateral kidney (n=139).Maximum diameter of calculi was 1.5 (0.8-2.6) cm.All patients underwent transperitoneal laparoscopic ureterolithtomy and double J stent was implanted intraoperatively.

Two cases were converted into open operation.Calculi moving up into pelvis occurred in 27 cases.And 25 cases converted into laparoscopic pyelolithotomy and stones were removed successfully.Two cases received extra extracorporeal shock wave lithotripsy postoperatively.The remaining 1142 cases were treated successfully with an operative duration of 56.1 (26-160) min and an estimated volume of blood loss at 45.2 (10-250) ml.The period of drainage tube was 3.1 (1-7) days.Postoperative hospitalization stay was 4.8 (3-9) days.One patient suffered urine leakage and healed at Day 6 days post-operation.Ten cases suffered D-J stent bladder non-arrival or retraction and double J stents were extracted by ureteroscopy.One case suffered paralytic ileus and recovered 7 days later.Another case suffered severe hematuria.No intestine or adjacent viscera injury was observed.The follow-up period of 827 cases was 7.3 (24-108) months.Among 12 cases of ureteral stricture, ureteral dilation (n=7) and ureteral anastosmosis (n=3) were performed.For 5 cases of renal atrophy, 3 underwent nephrectomy because of recurrent lumbago or persistent urinary infection.

Transperitoneal laparoscopic ureterolithotomy has the advantages of minimal morbidity, little postoperative discomfort and high stone clearance rate over open surgery.It should be widely adopted for the patients with upper ureteral impacted calculi.

To explore the clinical profiles of juvenile sudden sensorineural hearing loss (JSSNHL) and examine its clinical characteristics and prognosis.

A retrospective review was conducted for the clinical symptoms, audiological characteristics, hematological indices and prognosis in JSSNHL during the past 2 years (from June 2008 to November 2010).All patients were divided into 2 groups according to age, that is group childhood (A, 0-12 years old) and group adolescence (B, 13-18 years old).

JSSNHL patients were rarely associated with"aural fullness"symptoms.Two groups of patients with"tinnitus"symptom accounted for 88.2% and 89.5%.Those with"vertigo"symptom accounted for 47.1% and 44.4% respectively.Most patients (81.6%) showed severe and profound hearing loss.The most common types of audiometric curve were flat and total deafness.Some obvious differences existed between two groups in hematological indices, such as platelet count, concentrations of electrolyte ions, mean corpuscular volume and mean corpuscular hemoglobin.Almost half of them (42.1%) improved hearing level during systemic medical treatment.The patients of two groups showed no significant difference in efficacies.And the hearing enhancement degree of patients in group B was more apparent than that of group A.

JSSNHL has different clinical features in different age groups.And the outcomes of personalized treatment regimens may be further improved through classification and grading.

To evaluate the effects of molecular subtypes on the prognosis of breast cancer patients after BCT (breast-conserving therapy).

From January 1999 to December 2007, a total of 699 female patients on BCT were analyzed retrospectively.They were grouped according to the results of immunohistochemistry, they were grouped as Luminal A (ER+/PR+/HER2-, Ki-67<14%), Luminal B (ER+/PR+/HER2±, Ki-67>14%), HER2 (ER-/PR-/HER2+) and triple negative breast cancer (T NBC)(ER-/PR-/HER2-).

Among them, the recurrence rate, metastasis rate and survival rate within 5 years after BCT accounted for 6.0%, 6.0% and 91.8% respectively.There were statistical differences in metastasis rate and survival rate among patients with different molecular subtypes (P<0.001), lymph node status (P<0.001) and on chemotherapy or not (P<0.001) on multivariate analysis.Though, on univariate analysis, different molecular subtypes had different risks for local failure (P=0.022).On multivariate analysis, this difference was not statistically significant (P=0.081).

Despite a worse prognosis of TNBC versus other subtypes, BCT is a viable option for selected TNBC patients.

To explore the clinical characteristics and prognosis of infantile-onset glycogen storage disease typeⅡ (GSDⅡ) in Chinese patients.

Sixteen children diagnosed as infantile-onset GSDⅡ in Shanghai Children′s Medical Center during Jan 2005 to Dec 2012 were recruited.Their disease history, presenting symptom, physical signs, biochemical tests and examinations of electrocardiogram and echocardiography were analyzed retrospectively.Follow-up data on motor development and survival were also collected and analyzed retrospectively.

16 cases were diagnosed as infantile-onset GSDⅡ (10 males,6 females), in which the peripheral blood levels of acidic α-glucosidase were remarkably low or completely absent.All of them were complicated with cardiac hypertrophy and left ventricular mass index was 161-616 g/m². Severe muscular weakness, hypotonia and development lag were found in all during the follow-up.Creatine kinase was detected in 15 patients and its level became significantly elevated in 14 of them.Alanine aminotransferase and aspartate aminotransferase were detected in 15 patients and their levels became significantly elevated in all of them.The median age was 3.6 (2.0-6.8) months at symptom onset and 6.5 (3.8-9.3) months at diagnosis.And 14 of them died during the follow-up and the median age at death was 9.0 (4.7-18.7) months.

As a fatal disease, infantile-onset GSDⅡ has the prominent clinical manifestations of progressive cardiac hypertrophy and muscular weakness or hypotonia.The clinical features and nature history of Chinese patients are similar as those reported in other countries.Detection of acidic α-glucosidase activity in peripheral blood is an effective way of screening for infantile-onset GSDⅡ.

To preliminarily assess the prognosis of patients with multiple organ dysfunction syndrome (MODSE) and analyze their influencing factors.

The clinical data of 365 MODSE patients admitted into Chinese PLA General Hospital during January 2009 to June 2012 were analyzed retrospectively.According to 28-day outcomes, they were divided into 2 groups (28-day survival and non-survival) while 4 groups according to age.Then these prognosis were evaluated with the current scoring systems ( (acute physiology and chronic health evaluationⅡ (APACHEⅡ&Ⅲ), sample acute physiological score (SAPSⅡ) and multiple organ dysfunction score (MODS) ).The predictive powers were compared by receiver operating characteristic (ROC) curve.Finally a binary logistic regression analysis was performed to evaluate the relevant influencing prognostic factors of MODSE.

The mean age was (77.8±9.1) years, mean number of failed organs (3.6±1.2) and a 28-day mortality 45.8%.The ages of non-survival group were older than those of survival group ((78.1±9.1) vs (76.7±11.0) years).The number of organ failures ((4.3±1.1) vs (3.1±1.0) ) and scores (APACHEⅡ: (28±7) vs (20±8), APACHEⅢ: (106±27) vs (75±31),SAPSⅡ: (64±16) vs (46±18), MODS: (9±3) vs (6±3) ) of non-survivals were significantly higher than that of survivals.The area under ROC curve of these four score systems were 0.790, 0.781, 0.780 and 0.780 respectively.Compared to the above systems, SAPSⅡhad the best performance in sensitivity while APACHEⅡwas more valuable in specificity.All clinical data underwent binary logistic regression and the results showed that plasma concentration of albumin and mean arterial pressure (MAP) offered beneficial outcomes while age and number of organ failures had unfavorable prognosis.The greater patient age, the higher their mortality.

All four scoring systems have accurate prognostic predications of MODSE patients.And the predictive power of APACHEⅡis the best.Plasma concentration of albumin, MAP, age and organ failure number are independent prognostic factors in MODSE patients.

To explore the shift of Th2/Th1 type cytokine profiles in middle ear and peripheral blood of chronic otitis media with effusion (COME) patients.

Middle ear effusions (MEEs) and peripheral bloods were collected from 30 COME patients between April 2011 and July 2012 at Department of Otolaryngology, Beijing Tongren Hospital, Capital Medical University.Peripheral blood samples were collected from 20 healthy controls.The expressions of IFN-γ (Th1 cytokine) and IL-4 (Th2 cytokine) in peripheral blood and middle ear fluid were assessed by flow cytometry and cytometric bead array.The expression levels of cytokines in peripheral blood were compared between patient and control groups.Moreover, the levels of cytokines in the Middle ear fluid and the supernatant of COME patients were also compared.

Compared to the control group, the level of IL-4 in peripheral blood of COME patients significantly increased (0.7%±0.8% vs 2.4%±4.5%,P<0.05).The ratio of IFN-γ positive cells/IL-4 positive cells was significantly lower in peripheral blood of COME patients than that of control group (17±13 vs 35±19, P<0.05).However, the level of IFN-γshowed no significant difference between two groups (12%±8% vs 13%±8%, P>0.05).The levels of IL-4 and IFN-γin middle ear fluid and the supernatant of the case group showed no significant difference.

COME may be a Th2 predominant disease.Th1/Th2 imbalance is probably involved in the pathogenesis of COME.The role of the allergic reaction in middle ear microenvironment remains uncertain.

To explore the mathematics cognitive function of children with attention deficit hyperactivity disorder and explore neural mechanisms with event-related potential (ERP) and behaviors.

Behavior data and ERPelicited by performing mental calculation tasks were recorded in 27 children with ADHD and 29 normal controls from July to October 2012 at Third Affiliated Hospital of Soochow University.The differences of behaviors and N2 component of ERP were compared and analyzed.

The reaction time of the children with ADHD were longer than the control group in addition,subtraction and multiplication ((949±144) vs (829±166) ms, (981±129) vs (856±170) ms, (944±136) vs (825±172) ms, all P<0.05).While the correct rate were less than normal control in all three arithmetic operations (0.80% (0.72%, 0.88%) vs 0.90% (0.85%,0.96%),0.78% (0.64%,0.85%) vs 0.90% (0.84%,0.93%), 0.86% (0.74%,0.92%) vs 0.93% (0.90%,0.98%),all P<0.05).N2 component could be elicited by all subjects in forehead.The amplitude of N2 of children with ADHD were significantly lower than control group in all three arithmetic operations at left frontal (F3: (-3.5±5.2) vs (-6.7±3.5) μV, (-3.8±4.0) vs (-7.4±4.5) μV,-5.8 (-7.6,1.6) vs -6.4 (-10.3,-4.9) μV, all P<0.05) and Fz ((-4.3±6.4) vs (-7.4±4.2) μV, (-5.0±5.4) vs (-7.9±4.6) μV,-5.2 (-9.7,-0.6) vs-7.9 (-10.5,-5.1) μV, all P<0.05),the latency of ADHD group were prolonger than controls in subtraction operations at right and left frontal ((328±36) vs (307±27) ms, 325 (307,354) vs 309 (280, 330) ms) and frontal electrodes ((331±35) vs (311±30) ms, all P<0.05).In addition and multiplication operations, there was no significant difference in latency (all P>0.05).

The children with ADHD have weak capacities of inhibition irrelevant information and paying attention to control.Their deficits in mental arithmetics may be due to the difficulties of selecting the best strategy during cognitive tasks.

To explore the relationship between different tissue levels of polyunsaturated fatty acid (PUFA) and the clinicopathologic parameters in colorectal carcinoma (CRC) and evaluate their prognostic significance.

Fresh frozen malignant tissue was obtained from 82 colorectal cancer patients at PL A general Hospital from December 2010 to March 2011.The immunohistochemical results were obtained for vascular endothelial growth factor (VEGF), P53 and Ki-67.The relationship between the PUFA level and such clinicopathological profiles as age, gender, location, differentiation degree, TNM (tumor, node and metastasis) stage, VEGF, Ki-67 and P53 was analyzed.

Tissue level of arachidonic acid (AA) in patients aged over 60 years (n=44) was significantly lower than those under 60 years (n=38) (0.12%±0.06% vs 0.17%±0.09%, P=0.045).In patients with tumor size<5 cm (n=42), tissue level of EPA was significantly higher (0.29%±0.13% vs 0.20%±0.14%, P=0.030) whileω-6/ω-3 PUFA lower (10.8±2.6 vs 13.2±6.4, P=0.031).Significant statistical difference existed in tissue level of LA, AA/ω-3 PUFA in different differentiation degrees (P=0.013, 0.027).Tissue level of linoleic acid (LA) in poorly differentiated tumor was the highest (19.9%±6.3%) while AA/ω-3 PUFA the lowest (4.1±2.0, P<0.05).Tissue level of LA was higher in VEGF-positive tumors than those in VEGFnegative counterparts (16.2%±3.7% vs 13.9%±2.7%, P=0.009) while the ratios AA/ω-3 PUFA, AA/ω-6 PUFA, AA/LA in VEGF-positive tumors were lower than those in VEGF-negative counterparts (5.0±1.8 vs 6.7±3.3, 0.30±0.09 vs 0.34±0.09, 0.50±0.21 vs 0.61±0.21, P=0.004, 0.038, 0.030).In Ki-67 negative LA was highest (22.5%±10.1%, P=0.048).No significant differences existed in the level of PUFA among the gender, the clinicopathological stage, lymph node metastasis and groups with differential expressions of P53 (all P>0.05).

The tissue levels of PUFA are somewhat correlated with the clinicopathologic parameters of CRC.And the prognosis of CRC may be evaluated through the test of PUFA.

To explore the therapeutic regimens of metastatic prostate cancer so as to provide the experimental rationales for its gene therapy.

The adenoviral vectors expressing cytokines interleukin-18 (IL-18) and interleukin-12 (IL-12) were used to induce tumor regression in a C57BL/6 murine model of prostate cancer (n=110).

Adenoviral vectors could express IL-18 and IL-12 effectively.The rates of tumorigenesis were 10/10, 10/10, 4/10, 5/10 and 2/10, the durations of tumor growth (12.3±1.5), (12.8±1.0), (15.4±1.3), (14.8±0.8), (24.5±2.2) days and the diameters of tumor nodule after inoculation 30 days (37.0±3.0), (35.0±4.6), (25.0±2.0), (27.0±4.1) and (9.5±3.2) mm respectively in inoculation wild-type, AdLacZ, AdmIL-18, Adh IL-12, AdmIL-18 and AdhIL-12 of RM-1 cell.Compared to the other four groups, AdmIL-18 and AdhIL-12 developed smaller and delayed tumors (P<0.01).Intratumoral injection of Adm IL-18 and Adh IL-12 could not only regress the established tumors, but also reduce the number of distal lung metastases (P<0.01).

Adenovirus-mediated delivery of IL-18 and IL-12 locally by intratumoral injection is highly effective in inducing specific antitumor immune responses.

To explore the anti-fibrotic effects of angiotensin (Ang)1-7 on bleomycin (BLM)-induced pulmonary fibrosis in rats.

Eighteen Wister male rats were randomly divided into 3 groups,including control group (intratracheal instillation with physiological saline and subcutaneous micropump with bi-distilled water at the rate of 0.29μl/h), BLM group (intratracheal instillation with bleomycin and subcutaneous micro-pump with bi-distilled water at the same rate) and BLM+ Ang1-7 group (intratracheal instillation with bleomycin and subcutaneous micro-pump with Ang1-7 at a dose of 25μg·kg^-1·h^-1 at the same rate).At Day 28, lung tissues were collected.Histological changes of lungs were evaluated by hematoxylin and eosin and Masson′s trichrome stains.Collagen content of lung tissues was assessed by hydroxyprolin concentration.Then the products of protein and RNA were collected.And Western blot and realtime polymerase chain reaction (RT-PCR) were used to detect the protein or mRNA of TGF-β1 andα-collagenⅠ.Human embryonic lung fibroblast (HFL-1) was divided into 5 groups: (1) control group:no stimulation; (2) AngⅡgroup: stimulation of AngⅡ (10^-7 mol/L); (3) Ang1-7 group: stimulation of Ang1-7 (10^-7mol/L); (4) Ang1-7 plus AngⅡgroup: stimulation by AngⅡ (10^-7mol/L) with Ang1-7 (10^-7mol/L) pre-treatment; (5) Ang1-7+ AngⅡ+ A-779 group: stimulation by AngⅡand Ang1-7 (10^-7mol/L) with Mas receptor inhibitor A-779 (10^-6mol/L) pre-treatment.Then the products of protein and RNA were collected.And QuantiGene and RT-PCR were used to detect the activation of TGF-β1,andα-collagenⅠmRNA.

Compared with control group, fibrosis score and hydroxyproline concentrations increased significantly in BLM group, but declined in BLM+ Ang1-7 group.The difference was statistically significant (P<0.05).TGF-β1 mRNA,α-collagen Ⅰ mRNA andα-collagen Ⅰ protein level were up-regulated by BLM (4.45±0.45 vs 1.00±0.20,5.14±0.55 vs 1.00±0.08,1.48±0.34 vs 0.23±0.11)(all P<0.05); while compared with BLM group, those of BLM+ Ang1-7 group were downregulated (2.80±0.35, 3.10±0.52, 0.49±0.11)(all P<0.05).In vitro: TGF-β1 mRNA andα-collagenⅠmRNA level were up-regulated by AngⅡ (1.67±0.26 vs 1.00±0.10, 4.86±1.36 vs 1.46±0.54) (all P<0.05); while those of AngⅡ+ Ang1-7 group were down-regulated (0.91±0.30, 1.57±0.27) compared with AngⅡ group (all P<0.05); no significant difference existed between the AngⅡ+ Ang1-7+A-779 group (1.25±0.14, 1.29±0.49) and AngⅡ+ Ang1-7 group (P>0.05).

Ang1-7 has anti-fibrous effect upon bleomycin-induced pulmonary fibrosis in rats andsuch an effect of Ang1-7 may be associated with AngⅡ-induced expression of TGF-β1.