The effect of early radiofrequency turbinate reduction, intranasal steroid, and antihistamine H-1 on persistent allergic rhinitis: a randomized clinical trial

Abstract Objective: We aimed to evaluate the effect of radiofrequency turbinate reduction as an initial treatment on clinical improvement, inflammatory mediators, and remodeling process. Methods: Between July 2018- February 2020, 32 patients with moderate-severe persistent AR were randomly divided into 2 groups. Intervention group received radiofrequency turbinate reduction followed by intranasal steroid and Antihistamine H-1 (AH-1), control group received intranasal steroid and AH-1. Both groups were evaluated for clinical improvement (using visual analogue scale based on total nasal symptoms score, peak nasal inspiratory flow, and turbinate size using imageJ) after 4 and 8 weeks of treatment. Inflammatory mediators (ELISA from nasal secretions was performed to measure ECP, IL-5, and HSP-70) and remodeling markers (nasal biopsy followed by immunohistochemistry examination was performed to evaluate MMP-9, TIMP-1, and PAI-1) were evaluated in week 4. Results: Three patients dropped out of the study, resulting in 16 patients in intervention group and 13 patients in control group. At week 4, clinical response improved significantly in the intervention group compared to control group (Chi-Square test, p<0.05). Compared to control, intervention group experienced a reduction of IL-5 and no significant change in ECP level (Mann Whitney test, p>0.05). Reduction in the ratio of MMP-9/TIMP-1 were significantly higher in intervention group (unpaired t-test, p< 0,05). Meanwhile, increase in HSP-70 in the intervention group was slightly lower than in control group, but the difference with control group was not significant (Mann Whitney test, p>0.05). Conclusion: Early radiofrequency turbinate reduction followed by pharmacotherapy given to persistent moderate-severe AR patients give more improvement only in early clinical symptoms and reduce MMP-9/TIMP-1 ratio, thus it might be suggested as one of the adjuvant therapies for the management of moderate-severe persistent AR. However, further investigation with a larger sample size and longer follow-up period is needed. Level of evidence: 1B.

Captopril inhibits thioacetamide-induced chronic liver injury associated with the suppression of inflammation / hypoxia- inducible factor 1-alpha / profibrogenic axis-mediated hepatotoxicity in rats / Captopril inhibe la lesión hepática crónica inducida por tioacetamida asociada con la supresión de la inflamación/factor 1-alfa inducible por hipoxia/hepatotoxicidad mediada por el eje profibrogénico en rata

SUMMARY: Liver transplantation is the only available method to treat liver failure induced by chronic liver injury. We sought to determine whether the angiotensin-converting enzyme inhibitor, captopril, can inhibit the development of chronic liver injury induced by the hepatotoxic agent thioacetamide (TAA) in association with the suppression of inflammation (hsCRP, TNF-α, and IL-6) / hypoxia- inducible factor 1-alpha (HIF-1α) / profibrosis (TIMP-1, MMP-9, and α-SMA) axis that mediates liver injury. Therefore, the model group of rats was injected for eight weeks with 200 mg/kg TAA starting at week two. The protective group was pretreated with 150 mg/ kg captopril daily for two weeks prior to TAA injections and continued receiving both capropril and TAA agents until being humanely scrificed at week 10. We observed a substantial damage to liver tissue in the model group as demonstrated by a significant (p<0.0001) increase in blood and hepatic tissue levels of high sensitivity C-reactive protein (hsCRP), tumor necrosis factor-a (TNF-α), interleukin- 6 (L-6), HIF-1α, tissue inhibitor of metalloproteinases-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), alpha-smooth muscle actin (α-SMA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). All these parameters were significantly (p<0.0244) protected by captopril. Also, a significant (p<0.0001) positive correlation was observed between a-SMA (profibrosis) and the serum and tissue levels of hsCRP, TNF-α, HIF-1α, TIMP-1, MMP-9, and ALT. Thus, these findings suggest that the induction of chronic liver injury by the hepatotoxic compound, TAA is associated with the upregulation of inflammation/HIF-1α/profibrosis, with captopril exhibiting beneficial hepatic pleotropic effects.

El trasplante de hígado es el único método disponible para tratar la insuficiencia hepática inducida por una lesión hepática crónica. Buscamos determinar si el inhibidor de la enzima convertidora de angiotensina, captopril, puede inhibir el desarrollo de lesión hepática crónica inducida por el agente hepatotóxico tioacetamida (TAA) en asociación con la supresión de la inflamación (hsCRP, TNF-α e IL-6) / factor inducible por hipoxia 1-alfa (HIF-1α) / profibrosis (TIMP-1, MMP-9 y α- SMA) eje que media la lesión hepática. Por lo tanto, al grupo modelo de ratas se le inyectó durante ocho semanas 200 mg/kg de TAA a partir de la semana dos. El grupo protector fue pretratado con 150 mg/kg de captopril al día durante dos semanas antes de las inyecciones de TAA y continuó recibiendo capropril y agentes TAA hasta que fue sacrificado en la semana 10. Observamos un daño sustancial en el tejido hepático en el grupo modelo, como lo demuestra un aumento significativo (p<0,0001) de los niveles en sangre y tejido hepático de proteína C reactiva de alta sensibilidad (hsCRP), factor de necrosis tumoral-α (TNF-a), interleucina-6 (L-6), HIF-1α, inhibidor tisular de metaloproteinasas-1 (TIMP-1), metaloproteinasa de matriz-9 (MMP-9), actina de músculo liso alfa (α-SMA), alanina aminotransferasa (ALT) y aspartato aminotransferasa (AST). Todos estos parámetros estaban significativamente (p<0,0244) protegidos por captopril. Además, se observó una correlación positiva significativa (p<0,0001) entre α-SMA (profibrosis) y los niveles séricos y tisulares de hsCRP, TNF-α, HIF-1α, TIMP- 1, MMP-9 y ALT. Por lo tanto, estos hallazgos sugieren que la inducción de daño hepático crónico por el compuesto hepatotóxico, TAA, está asociada con la regulación al alza de la inflamación/HIF-1α/profibrosis, con captopril exhibiendo efectos pleotrópicos hepáticos beneficiosos.

Role of PI3K/Akt/mTOR pathway-mediated macrophage autophagy in affecting the phenotype transformation of lung fibroblasts induced by silica dust exposure / 中南大学学报(医学版)

OBJECTIVES@#The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy. Autophagy plays a key role in the formation of silicosis fibrosis. The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis. This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy.@*METHODS@#The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h. Macrophages were exposed to different concentrations (0, 25, 50, 100, 200, 400 μg/mL) and different times (0, 6, 12, 24, 48 h) of SiO2 dust suspension. The survival rate of macrophages was measured by cell counting kit-8 (CCK-8) method. Enzyme linked immunosorbent assay (ELISA) was used to measure the contents of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) in the cell supernatant. The co-culture system of macrophages and HFL-1 cells was established by transwell. A blank control group, a SiO2 group, a LY294002 group, a SC79 group, a LY294002+SiO2 group, and a SC79+SiO2 group were set up in this experiment. Macrophages in the LY294002+SiO2 group were pretreated with LY294002 (PI3K inhibitor) for 18 hours, and macrophages in the SC79+SiO2 group were pretreated with SC79 (Akt activator) for 24 hours, and then exposed to SiO2 (100 μg/mL) dust suspension for 12 hours. The expression of microtubule-associated protein 1 light chain 3 (LC3) protein in macrophages was detected by the immunofluorescence method. The protein expressions of PI3K, Akt, mTOR, Beclin-1, LC3 in macrophages, and collagen III (Col III), α-smooth muscle actin (α-SMA), fibronectin (FN), matrix metalloproteinase-1 (MMP-1), tissue metalloproteinase inhibitor-1 (TIMP-1) in HFL-1 cells were measured by Western blotting.@*RESULTS@#After the macrophages were exposed to SiO2 dust suspension of different concentrations for 12 h, the survival rates of macrophages were gradually decreased with the increase of SiO2 concentration. Compared with the 0 μg/mL group, the survival rates of macrophages in the 100, 200, and 400 μg/mL groups were significantly decreased, and the concentrations of TGF-β1 and TNF-α in the cell supernatant were obviously increased (all P<0.05). When 100 μg/mL SiO2 dust suspension was applied to macrophages, the survival rates of macrophages were decreased with the prolonged exposure time. Compared with the 0 h group, the survival rates of macrophages were significantly decreased (all P<0.05), the concentrations of TGF-β1 and TNF-α in the cell supernatant were significantly increased, and the protein expression levels of Beclin-1 and LC3II were increased markedly in the 6, 12, 24, and 48 h groups (all P<0.05). Immunofluorescence results demonstrated that after exposure to SiO2 (100 μg/mL) dust for 12 h, LC3 exhibited punctate aggregation and significantly higher fluorescence intensity compared to the blank control group (P<0.05). Compared with the blank control group, the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated in the SiO2 group (all P<0.05). Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were down-regulated and the protein expressions of LC3II and Beclin-1 were up-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were decreased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were down-regulated (all P<0.05) in the LY294002+SiO2 group. Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were up-regulated and the protein expressions of LC3II and Beclin-1 were down-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were increased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated (all P<0.05) in the SC79+SiO2 group.@*CONCLUSIONS@#Silica dust exposure inhibits the PI3K/Akt/mTOR pathway, increases autophagy and concentration of inflammatory factors in macrophages, and promotes the phenotype transformation of HFL-1 cells into myofibroblasts. The regulation of the PI3K/Akt/mTOR pathway can affect the autophagy induction and the concentration of inflammatory factors of macrophages by silica dust exposure, and then affect the phenotype transformation of HFL-1 cells into myofibroblasts induced by silica dust exposure.

Effects of blistering cupping combined with thunder-fire moxibustion on the efficacy and airway remodeling of cold-wheezing syndrome in bronchial asthma / 中国针灸

OBJECTIVE@#To investigate the clinical efficacy of the combined application of blistering cupping with thunder-fire moxibustion in treating bronchial asthma of cold-wheezing syndrome, and its influences on airway remodeling, inflammatory factors, lung function, and quality of life on the base of conventional western medicine treatment.@*METHODS@#A total of 76 patients with bronchial asthma of cold-wheezing syndrome were randomly divided into an observation group and a control group, 38 cases in each group. In the control group, the basic treatment was used, i.e. budesonide formoterol powder inhalation. In the observation group, on the basis of the treatment as the control group, blistering cupping combined with thunder-fire moxibustion was supplemented, Dazhui (GV 14), Danzhong (CV 17) and bilateral Feishu (BL 13), Gaohuang (BL 43), and Zhongfu (LU 1) were selected; blistering cupping was administered once a day and thunder-fire moxibustion was given twice a day. One course of treatment was composed of 7 days in both groups, and 2 courses of treatment were required. Before and after treatment, the airway remodeling indexes (matrix metalloproteinase-9 [MMP-9], tissue inhibitor of matrix metalloproteinase-1 [TIMP-1], and transforming growth factor-β1 [TGF-β1]) and inflammatory indexes (interleukin [IL] -1β、IL-25) were detected by using radioimmunoassay in the patients of the two groups. The lung function, traditional Chinese medicine symptom score, and asthma quality of life questionnaire (AQLQ) score were observed in the patients of the two groups.@*RESULTS@#After treatment, the serum levels of MMP-9, TIMP-1, TGF-β1, IL-1β, IL-25, peak expiratory flow (PEFR), traditional Chinese medicine symptom scores, and AQLQ scores were decreased compared with those before treatment in the patients of the two groups (P<0.05), and the results in the observation group were lower than those in the control group (P<0.05). After treatment, the first second forced expiratory volume (FEV1) and peak expiratory flow rate (PEF) were increased compared with those before treatment in the two groups (P<0.05), and the results in the observation group were higher than those in the control group (P<0.05).@*CONCLUSION@#On the basis of the conventional western medicine treatment, the combination of blistering cupping with thunder-fire moxibustion can effectively ameliorate the clinical symptoms of patients, reduce inflammatory levels, inhibit airway remodeling and improve the lung function and quality of life in the patients with bronchial asthma.

Qianjin Wenwu decoction suppresses renal interstitial fibrosis by enhancing the degradation of extracellular matrix in mice with unilateral ureteral obstruction / 中国天然药物

Diabetic kidney disease (DKD) is the most common complication of diabetes mellitus (DM). Qianjin Wenwu decoction (QWD), a well-known traditional Korean medicine, has been used for the treatment of DKD, with satisfactory therapeutic effects. This study was designed to investigate the active components and mechanisms of action of QWD in the treatment of DKD. The results demonstrated that a total of 13 active components in five types were found in QWD, including flavonoids, flavonoid glycosides, phenylpropionic acids, saponins, coumarins, and lignins. Two key proteins, TGF-β1 and TIMP-1, were identified as the target proteins through molecular docking. Furthermore, QWD significantly suppressed Scr and BUN levels which increased after unilateral ureteral obstruction (UUO). Hematoxylin & eosin (H&E) and Masson staining results demonstrated that QWD significantly alleviated renal interstitial fibrosis in UUO mice. We also found that QWD promoted ECM degradation by regulating MMP-9/TIMP-1 homeostasis to improve renal tubulointerstitial fibrosis and interfere with the expression and activity of TGF- β1 in DKD treatment. These findings explain the underlying mechanism of QWD for the treatment of DKD, and also provide methodological reference for investigating the mechanism of traditional medicine in the treatment of DKD.

Nanosilver alleviates foreign body reaction and facilitates wound repair by regulating macrophage polarization / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Foreign body reactions induced by macrophages often cause delay or failure of wound healing in the application of tissue engineering scaffolds. This study explores the application of nanosilver (NAg) to reduce foreign body reactions during scaffold transplantation. An NAg hybrid collagen-chitosan scaffold (NAg-CCS) was prepared using the freeze-drying method. The NAg-CCS was implanted on the back of rats to evaluate the effects on foreign body reactions. Skin tissue samples were collected for histological and immunological evaluation at variable intervals. Miniature pigs were used to assess the effects of NAg on skin wound healing. The wounds were photographed, and tissue samples were collected for molecular biological analysis at different time points post-transplantation. NAg-CCS has a porous structure and the results showed that it could release NAg constantly for two weeks. The NAg-CCS group rarely developed a foreign body reaction, while the blank-CCS group showed granulomas or necrosis in the subcutaneous grafting experiment. Both matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced significantly in the NAg-CCS group. The NAg-CCS group had higher interleukin (IL)-10 and lower IL-6 than the blank CCS group. In the wound healing study, M1 macrophage activation and inflammatory-related proteins (inducible nitric oxide synthase (iNOS), IL-6, and interferon-‍γ (IFN-‍γ)) were inhibited by NAg. In contrast, M2 macrophage activation and proinflammatory proteins (arginase-1, major histocompatibility complex-II (MHC-II), and found in inflammatory zone-1 (FIZZ-1)) were promoted, and this was responsible for suppressing the foreign body responses and accelerating wound healing. In conclusion, dermal scaffolds containing NAg suppressed the foreign body reaction by regulating macrophages and the expression of inflammatory cytokines, thereby promoting wound healing.

Changes of heparin-binding protein in severe burn patients during shock stage and its effects on human umbilical vein endothelial cells and neutrophils / 中华烧伤杂志

Objective: To investigate the changes of heparin-binding protein (HBP) in severe burn patients during shock stage and its effects on human umbilical vein endothelial cells (HUVECs) and neutrophils in vitro. Methods: Prospective observational and experimental research methods were used. Twenty severe burn patients who met the inclusion criteria and were admitted to the Department of Burns and Plastic Surgery of Affiliated Suzhou Hospital of Nanjing Medical University from August to November 2020 were included in severe burn group (12 males and 8 females, aged 44.5 (31.0, 58.0) years). During the same period, 20 healthy volunteers with normal physical examination results in the unit's Physical Examination Center were recruited into healthy control group (13 males and 7 females, aged 39.5 (26.0, 53.0) years). Enzyme-linked immunosorbent assay (ELISA) method was used to detect the protein expression levels of HBP and tissue inhibitor of metalloproteinase 1 (TIMP-1) in plasma of patients within 48 hours after injury in severe burn group and in plasma of volunteers in healthy control group. The correlation between protein expression of HBP and that of TIMP-1 in the plasma in the two groups was analyzed by Pearson correlation analysis. The fourth passage of HUVECs in logarithmic growth phase were used for the experiment. The HUVECs were divided into normal control group with routine culture (the same treatment below) and recombinant HBP (rHBP)-treated 12 h group, rHBP-treated 24 h group, and rHBP-treated 48 h group with corresponding treatment according to the random number table (the same grouping method below), and the mRNA expression of TIMP-1 in cells was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction. The HUVECs were divided into normal control group and rHBP-treated 48 h group with corresponding treatment, and the protein expression of TIMP-1 in the cells was detected by Western blotting. The HUVECs were divided into normal control group, rHBP alone group, aprotinin alone group, and rHBP+aprotinin group treated with the corresponding reagents (with the final molarity of rHBP being 200 nmol/L and the final concentration of aprotinin being 20 μg/mL, respectively), cultured for 48 h, and ELISA was used to detect the protein expression of TIMP-1 in the culture supernatant of cells. The neutrophils were isolated from the peripheral venous blood of the aforementioned 10 healthy volunteers by immunomagnetic bead sorting, and the cells were divided into normal control group, recombinant TIMP-1 (rTIMP-1) alone group, phorbol acetate (PMA) alone group, and rTIMP-1+PMA group treated with corresponding reagents (with the final concentration of rTIMP-1 being 500 ng/mL and the final molarity of PMA being 10 nmol/L, respectively). After being cultured for 1 h, the expression of CD63 protein in cells was detected by immunofluorescence method, the positive expression rate of CD63 protein in cells was detected by flow cytometry, and the protein expression levels of HBP and myeloperoxidase (MPO) in the culture supernatant of cells were detected by ELISA. The normal control group underwent the above-mentioned related tests at appropriate time points. The number of samples was 3 in each group of cell experiment. Data were statistically analyzed with chi-square test, Mann-Whitney U test, Kruskal-Wallis H test, and Tamhane's T2 test. Results: The protein expression levels of HBP and TIMP-1 in the plasma of patients in severe burn group were 404.9 (283.1, 653.2) and 262.1 (240.6, 317.4) ng/mL, respectively, which were both significantly higher than 61.6 (45.0, 68.9) and 81.0 (66.3, 90.0) ng/mL of volunteers in healthy control group (with Z values of -5.41 and -5.21, respectively, P<0.01). The correlation between the protein expression of HBP and that of TIMP-1 in the plasma of volunteers in healthy control group was not strong (P>0.05). The protein expression of HBP was significantly positively correlated with that of TIMP-1 in the plasma of patients in severe burn group (r=0.64, P<0.01). Compared with that in normal control group, the mRNA expression of TIMP-1 in HUVECs was significantly increased in rHBP-treated 12 h group, rHBP-treated 24 h group, and rHBP-treated 48 h group (with t values of -3.58, -2.25, and -1.26, respectively, P<0.05). Western blotting detection showed that compared with that in normal control group, the protein expression of TIMP-1 in HUVECs in rHBP-treated 48 h group was significantly enhanced. After 48 h of culture, compared with that in normal control group, the protein expression level of TIMP-1 in the culture supernatant of HUVECs in rHBP alone group was significantly increased (t=9.43, P<0.05), while the protein expression level of TIMP-1 in the culture supernatant of HUVECs didn't change significantly in aprotinin alone group or rHBP+aprotinin group (P>0.05); compared with that in rHBP alone group, the protein expression level of TIMP-1 in the culture supernatant of HUVECs in rHBP+aprotinin group was significantly decreased (t=4.76, P<0.01). After 1 h of culture, the trend of CD63 protein expression in neutrophils detected by immunofluorescence method and that by flow cytometry were consistent in each group. After 1 h of culture, compared with that in normal control group, the positive expression rate of CD63 protein in the neutrophils and the protein expression levels of HBP and MPO in the culture supernatant of cells in rTIMP-1 alone group all had no significant changes (P>0.05), while the positive expression rate of CD63 protein in the neutrophils and the protein expression levels of HBP and MPO in the culture supernatant of cells were all significantly increased in PMA alone group and rTIMP-1+PMA group (with t values of 2.41, 3.82, 5.73, 1.05, 4.16, and 1.08, respectively, P<0.05 or P<0.01); compared with that in PMA alone group, the positive expression rate of CD63 protein in the neutrophils and the protein expression levels of HBP and MPO in the culture supernatant of cells in rTIMP-1+PMA group were all significantly decreased (with t values of 5.26, 2.83, and 1.26, respectively, P<0.05 or P<0.01). Conclusions: The expression level of HBP in the plasma of severe burn patients is increased during shock stage. HBP can induce HUVECs to secrete TIMP-1 in vitro, and TIMP-1 can reduce the expression of CD63 molecule in human neutrophils.

Effect of Taohong Siwu Decoction() early intervention on mesenchymal stem cells homing in fracture healing in rats / 中国骨伤

OBJECTIVE@#To observe the effects of Taohong Siwu Decoction(, THSWD) on the mesenchymal stem cells(MSCs) migration, homing number and cytokine expression in callus during the early process of fracture healing, and to explore the mechanism of THSWD on accelerationg fracture healing by regulating the homing of MSCs in rats.@*METHODS@#A rat model of right femoral shaft open fracture was established. Thirty-two 5-week-old male Sprague-Dawley rats, weighting 110 to 130 g, were divided into control group, low-dose group, medium-dose group and high-dose group by using random number table. Distilled water was given to the control group, and the other groups were given Taohong Siwu Decoction. The rats were gavaged twice a day for 5 consecutive days after surgery. Bone volume/tissue volume(BV/TV) and bone mineral density(BMD) were observed using micro-computed tomography (micro-CT) at 21 days after surgery. At 5 days post-fracture, peripheral blood MSCs from THSWD treated and untreated rats were cultured in vitro. Subsequently, the migration ability of MSCs was observed by cell migration assay. The number of MSCs homing to the callus at the early stage of fracture (5 d) was detected by Immunohistochemistry (IHC). Protein chip was used to detect the expression of cytokines in callus.@*RESULTS@#Micro-CT results showed that BV/TV was higher in the high-dose group than in the medium-dose group (P=0.032), and higher in the medium-dose group than in the low-dose group(P=0.041), with no difference between the control and low-dose group (P=0.651). In addition, there was no difference in BMD between low-dose group and the model group (P=0.671), and lower in the low-dose group than in the medium-dose group(P=0.018), and the medium-dose group was lower than the high-dose group(P=0.008). Cell migration assay showed that THSWD promotes enhanced the migration ability of peripheral blood MSCs. IHC assay revealed that CD45-, CD90+, CD29+ MSCs significantly increased in bone callus after THSWD intervention compared with the control group. Protein chip showed that THSWD promoted the upregulation of CINC-1(×2.91), CINC-3(×1.59), LIX(×1.5), Thymus Chemokine (×2.55), VEGF (×1.22) and the down-regulation of TIMP-1 (×2.98).@*CONCLUSION@#THSWD, a representative formula of "promoting blood circulation and removing blood stasis", can significantly accelerate fracture healing, and its mechanism may be related to enhancing the migration ability of peripheral blood MSCs and up-regulating CINC-1, CINC-3, LIX, Thymus Chemokine, VEGF and down-regulating TIMP-1 in bone callus, which promotes the peripheral blood MSCs homing in the early stage of fracture.

Overwork Affects Extracellular Matrix of Arterial Vessel Wall in Rats / 中国医学科学院学报

Objective To explore the effect of overwork (OW) on extracellular matrix of arterial vessel wall in rats. Methods Random number grouping method was employed to assign 18 Sprague-Dawley rats into three groups(n=6):the control group(no special treatment),group OW(forced swimming twice a day for 15 days),and sleep deficiency(SD)+OW group(in addition to forced swimming twice a day,the rats were put on the platforms in water to limit sleep for 15 days).On the 16th day,the abdominal aorta and common carotid artery were collected after blood sampling from heart under deep anesthesia.A part of the abdominal aorta sample was taken for Masson staining of collagen fiber,and Verhoeff-Van Gieson staining was carried out for the elastic fiber of common carotid artery.Image J was employed for the quantitative analysis of collagen fiber and elastic fiber content.The expression of collagen 1(Col-1) protein was quantified by immunohistochemistry and the ultrastructure of vascular matrix was examined by transmission electron microscopy.The other part of the abdominal aorta sample was used to determine the mRNA levels of matrix metalloproteinase(MMP)-1,MMP-2,MMP-9,tissue inhibitor of metalloproteinases-1(TIMP-1),and Col-1 by quantitative real-time polymerase chain reaction. Results Compared with that in control group,the content of collagen fiber in groups OW and SD+OW had no significant change(all P>0.05);the content of elastic fiber in groups OW and SD+OW decreased(all P<0.001) and had no significant difference between each other(P>0.05).The vascular vessel wall of group OW showed slight fiber breakage,while that of group SD+OW presented wormhole-like or spongy fiber fragmentation.The mRNA levels of MMP-1 and MMP-2 in groups OW and SD+OW had no significant difference between each other(P>0.05) but were higher than that in control group(all P<0.001).The mRNA levels of MMP-9 and TIMP-1 had no significant difference among the three groups(all P>0.05).Groups OW and SD+OW had lower mRNA level(all P<0.001) and protein level(all P<0.001) of Col-1 than control group,while the mRNA and protein levels of Col-1 had no significant difference between groups OW and SD+OW(P>0.05). Conclusion OW can reduce the content of Col-1 and elastic fibers in the extracellular matrix of arterial vessels,destroy the elastic lamina of vascular wall,up-regulate the expression of MMP-1 and MMP-2,thereby injuring arterial vessels.

In vitro homeostasis of rat oral epithelial cell cultures following withdrawal of periodontal pathogens

Abstract Inflammation of periodontal tissues is the consequence of interaction between periodontal pathogens and immune system. This is associated with increased expression of inflammatory cytokines, which may exert destructive effect to the periodontal tissues when released over long period. The aim of this study was to chronologically track the homeostasis of oral keratinocytes following removal of periodontal pathogens. This was done by investigating expression of selected inflammatory markers and integrity of epithelial monolayers in vitro. Rat oral keratinocytes were stimulated with heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis over 7-days then bacteria were washed away and epithelial cells re-cultured for 3-days. Expression of IL-1β, IL-6, and IL-8 was measured by ELISA while transcription of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase -8 (MMP-8) was measured by polymerase chain reaction before and after removal of bacteria. Integrity of epithelial sheet was investigated by using transepithelial electrical resistance. Data showed general downregulation of IL-1b, IL-6, and IL-8 associated with restoring transcription of TIMP-1 and MMP-8 to normal level following removal of bacteria from epithelial cultures. However, expression of IL-8 and MMP-8 remained significantly higher than unstimulated epithelial cells despite withdrawal of F. nucleatum and P. gingivalis respectively from oral keratinocytes cultures. In addition, integrity of epithelial barrier function remained compromised even after removal of P. gingivalis. Results suggest that even after three days following removal of periodontal pathogens, oral keratinocytes sustained persistent upregulation of certain inflammatory markers that could compromise integrity of epithelial barrier function.

Resumo A inflamação dos tecidos periodontais é a consequência da interação entre patógenos periodontais e o sistema imunológico. Isso está associado ao aumento da expressão de citocinas inflamatórias, que podem exercer efeito destrutivo nos tecidos periodontais quando liberadas por um longo período. O objetivo deste estudo foi rastrear cronologicamente a homeostase dos queratinócitos orais após a remoção dos patógenos periodontais. Isto foi feito através da investigação da expressão de marcadores inflamatórios selecionados e da integridade de monocamadas epiteliais in vitro. Os queratinócitos orais de rato foram estimulados com Fusobacterium nucleatum e Porphyromonas gingivalis destruídas pelo calor por 7 dias, depois as bactérias foram lavadas e as células epiteliais foram cultivadas novamente por 3 dias. A expressão de IL-1b, IL-6 e IL-8 foi medida por ELISA, enquanto a transcrição do inibidor tecidual de metaloproteinase-1 (TIMP-1) e matriz metalopeptidase-8 (MMP-8) foi medida por reação em cadeia da polimerase antes e após a remoção de bactérias. A integridade da folha epitelial foi investigada usando resistência elétrica transepitelial. Os dados mostraram uma regulação negativa geral de IL-1b, IL-6 e IL-8 associada à restauração da transcrição de TIMP-1 e MMP-8 para o nível normal após a remoção de bactérias de culturas epiteliais. No entanto, a expressão de IL-8 e MMP-8 permaneceu significativamente maior que as células epiteliais não estimuladas, apesar da retirada de F. nucleatum e P. gingivalis, respectivamente, das culturas de queratinócitos orais. Além disso, a integridade da função da barreira epitelial permaneceu comprometida mesmo após a remoção de P. gingivalis. Os resultados sugerem que, mesmo após três dias após a remoção dos patógenos periodontais, os queratinócitos orais sustentaram uma regulação positiva persistente de certos marcadores inflamatórios que poderiam comprometer a integridade da função da barreira epitelial.

Levels of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 are related to cardiopulmonary injury in fetal inflammatory response syndrome

OBJECTIVES: To evaluate the diagnostic value of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and the MMP-9/TIMP-1 ratio in fetal inflammatory response syndrome (FIRS), and determine a possible association with the incidence of bronchopulmonary dysplasia (BPD) and myocardial injury. METHODS: Overall, 61 cases of preterm infants with FIRS were divided into the FIRS group 1 (≤32 weeks) and FIRS group 2 (32 to 37 weeks). Similarly, 57 cases of normal preterm infants were divided into Control group 1 and Control group 2. Levels of interleukin-6 (IL-6), MMP-9, and TIMP-1 were detected by enzyme-linked immunosorbent assay. Spearman's linear correlation was used to analyze the relationship between dependent variables. Pathological changes were examined by hematoxylin and eosin (HE) staining and in amniotic fluid smears. RESULTS: Levels of IL-6, MMP-9, and TIMP-1, and the MMP-9/TIMP-1 ratio were significantly higher in the FIRS group than in the Control groups. IL-6 was positively correlated with MMP-9, TIMP-1, and the MMP-9/TIMP-1 ratio. Areas under the curve (AUC) of MMP-9, TIMP-1, and the MMP-9/TIMP-1 ratio were 0.92, 0.90, and 0.95, respectively. HE staining and amniotic fluid smears showed the aggregation of inflammatory cells. MMP-9, TIMP-1, and the MMP-9/TIMP-1 ratio were closely related to the incidence of BPD (≤32 weeks) and myocardial injury (<37 weeks) in preterm infants. CONCLUSION: MMP-9, TIMP-1, and the MMP-9/TIMP-1 ratio revealed a certain diagnostic value for FIRS; combined with gestational age, these parameters were effective for predicting cardiopulmonary injury.

The Identification of Immune-Related Plasma Proteins Associated with Spontaneous Preterm Delivery and Intra-Amniotic Infection in Women with Premature Cervical Dilation or an Asymptomatic Short Cervix

BACKGROUND: We aimed to investigate whether various immune-related plasma proteins, alone or in combination with conventional clinical risk factors, can predict spontaneous preterm delivery (SPTD) and intra-amniotic infection in women with premature cervical dilation or a short cervix (≤ 25 mm).METHODS: This retrospective study included 80 asymptomatic women with premature cervical dilation (n = 50) or a short cervix (n = 30), who underwent amniocentesis at 17–29 weeks. Amniotic fluid (AF) was cultured, and maternal plasma was assayed for interleukin (IL)-6, matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinases (TIMP)-1, and complements C3a and C5a, using enzyme-linked immunosorbent assay (ELISA) kits. The primary outcome measures were SPTD at < 32 weeks and positive AF cultures.RESULTS: The plasma levels of IL-6, C3a, and C5a, but not of MMP-9 and TIMP-1, were significantly higher in women with SPTD at < 32 weeks than in those who delivered at ≥ 32 weeks. The women who delivered at < 32 weeks had more advanced cervical dilatation, and higher rates of antibiotic and tocolytic administration and were less likely to be given vaginal progesterone than those who delivered at ≥ 32 weeks. Using a stepwise regression analysis, a combined prediction model was developed, which included the plasma IL-6 and C3a levels, and cervical dilatation (area under the curve [AUC], 0.901). The AUC for this model was significantly greater than that for any single variable included in the predictive model. In the univariate analysis, plasma IL-6 level was the only significant predictor of intra-amniotic infection.CONCLUSION: In women with premature cervical dilation or a short cervix, maternal plasma IL-6, C3a, and C5a levels could be useful non-invasive predictors of SPTD at < 32 weeks. A combination of these biomarkers and conventional clinical factors may clearly improve the predictability for SPTD, as compared with the biomarkers alone. An increased plasma level of IL-6 predicted intra-amniotic infection.

Follistatin Mitigates Myofibroblast Differentiation and Collagen Synthesis of Fibroblasts from Scar Tissue around Injured Flexor Tendons

Effect of Relaxin Expression from an Alginate Gel-Encapsulated Adenovirus on Scar Remodeling in a Pig Model

PURPOSE: Relaxin (RLX) is a transforming growth factor-β1 (TGF-β1) antagonist that is believed to function as a potent collagen re-arranger and a major suppressor of extracellular matrix components. Adenoviruses (Ads) are accepted vectors for cancer gene therapy. However, repeated treatments of Ad are limited by short-term biological activity in vivo. The efficacy of sustained RLX expression to scar remodeling was assessed using an injectable alginate gel-matrix system. MATERIALS AND METHODS: Pig scar tissue was treated with relaxin-expressing Ad loaded in alginate gel (gel/Ad-RLX). Surface areas, color, and pliability of scars were compared, and various factors influencing scar formation and collagen arrangement were analyzed. RESULTS: Gel/Ad-RLX decreased scar size, color index, and pliability. Immunohistochemistry showed decreased levels of major extracellular matrix proteins in the gel/Ad-RLX-treated group. Furthermore, treatment with gel/Ad-RLX reduced expression of tissue inhibitor of metalloproteinase-1 and alpha-smooth muscle actin and markedly increased expression of matrix metalloproteinase-1 in pig scar tissues. Gel/Ad-RLX also significantly downregulated TGF-β1 and upregulated TGF-β3 mRNAs in pig scar tissues. CONCLUSION: These results support a prominent role for RLX in scar remodeling and suggest that gel/Ad-RLX may have therapeutic effects on scar formation.

Notum attenuates HBV-related liver fibrosis through inhibiting Wnt 5a mediated non-canonical pathways

BACKGROUND: Non-canonical Wnt pathways play important roles in liver fibrosis. Notum is a newly discovered inhibitor to Wnt proteins. This study was to investigate anti-fibrotic effects of Notum. METHODS: 53 patients with hepatitis B virus (HBV) infection as well as a cell co-culture system of LX-2 and Hep AD38 cells were engaged in this study. Clinical, biological and virological data of each patient were analyzed. Cell viability was detected at different time points. mRNA and protein levels of NFATc1 (Nuclear factor of activated T-cells), Jnk, α-SMA, Col1A1 and TIMP-1 were detected both in LX-2 and liver tissue. Protein levels of NFATc1 and Jnk in liver tissue and their correlations with fibrosis score were analyzed. RESULTS: Hepatitis B virus replication up-regulated Wnt5a induced NFATc1 and Jnk activity in Hep AD38. Notum suppressed NFATc1, Jnk and fibrosis genes expression, reduced cell viability in co-cultured LX-2 cells induced by HBV. Interestingly, Patients with HBV DNA > 5log copies/ml had higher mRNA levels of NFATc1 and fibrosis genes than patients with HBV DNA < 5log copies/ml. Most importantly, protein expressions of NFATc1 and pJnk have positive correlations with liver fibrosis scores in HBV-infected patients. CONCLUSIONS: Our data showed that Notum inhibited HBV-induced liver fibrosis through down-regulating Wnt 5a mediated non-canonical pathways. This study shed light on anti-fibrotic treatment.

Coexistence of proangiogenic potential and increased MMP-9, TIMP-1, and TIMP-2 levels in the plasma of patients with critical limb ischemia / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The objective of this study was to assess the angiogenic potential expressed as a quotient of vascular endothelial growth factor A (VEGF-A), as an indicator of proangiogenic activity, and the circulating receptors (soluble VEGF receptor protein R1 (sVEGFR-1) and sVEGFR-2), as indicators of the effect of angiogenic inhibition, depending on the concentrations of matrix metalloproteinase 2 (MMP-2) and MMP-9 and their tissue inhibitor 1 (TIMP-1) and TIMP-2 in the plasma of patients with lower extremity artery disease (LEAD). These blood parameters in patients with intermittent claudication (IC) and critical limb ischemia (CLI) were compared for select clinical and biochemical features. Stimulation of angiogenesis in the plasma of individuals with LEAD was evident as indicated by the significant increase in VEGF-A concentration along with reduced inhibition depending on circulating receptors sVEGFR-1 and sVEGFR-2. Critical ischemia was associated with higher VEGF-A, MMP-9, TIMP-1, and TIMP-2 concentrations than in the case of IC.

Expressions of TOLL-like receptor 4 (TLR-4) and matrix metalloproteinase 9 (MMP-9)/Tissue inhibitor of metalloproteinase 1 (TIMP-1) in pulmonary blood vessels with chronic obstructive pulmonary diseases and their relationships with pulmonary vascular remodelling

SUMMARY OBJECTIVE: This study aims at investigating the expressions of TOLL-like receptor 4 (TLR-4) and matrix metalloproteinase 9 (MMP-9)/ tissue inhibitor of metalloproteinase 1 (TIMP-1) in pulmonary blood vessels with chronic obstructive pulmonary disease (COPD) and their relationships with pulmonary vascular remodelling (PVR). METHODS: 60 para-tumour tissues were divided into the COPD group and the control group (n=30); the inflammations, pulmonary artery wall area/total artery area (WA%), and wall thickness/vascular outer diameter (WT%) were compared. The expressions of TLR-4, MMP-9/TIMP-1, and PCNA in pulmonary vascular smooth muscle cells were detected, and their relationships with PVR were then analysed. RESULTS: The inflammations (1.6±0.8), WA% (44.0±6.4), and WT% (27.3±3.3) in the COPD group were higher than in the control group (0.3±0.5, 26.1±2.8, 15.6±1.8), and the expressions of TLR-4 (31.4±147) and MMP-9/TIMP-1 (2.2±2.6) were increased compared to the control group (4.7±4.5, 1.9±12). Correlation analysis: TLR-4 and MMP-9/TIMP-1 were positively correlated with the inflammations (r=0.18, P<0.01), WA% (r=0.68, P<0.01), and WT% (r=0.73, P<0.01), as well as positively correlated with the expression of PCNA (r=0.44, P<0.01); the upregulation of TLR-4 was positively correlated with the expressions of MMP-9 and TIMP-1. CONCLUSIONS: The upregulation of TLR-4 in the pulmonary arterial smooth muscle cells of COPD patients could promote the inflammations and the MMP-9 expression, thus causing abnormal degradation of extracellular matrix, so it played an important role in the process of PVR.

RESUMO OBJETIVO: Este estudo tem como objetivo investigar as expressões de TOLL-like receptor 4 (TLR-4) e metaloproteinase 9 da matriz (MMP-9)/inibidor de tecido da metaloproteinase 1 (TIMP-1) em vasos sanguíneos pulmonares com doença pulmonar obstrutiva crônica (DPOC) e suas relações com o remodelamento vascular pulmonar (PVR). MÉTODOS: Sessenta tecidos paratumorais foram divididos em grupo COPD e o grupo controle (n = 30). Foram comparadas as inflamações, área da parede da artéria pulmonar/área da artéria total (WA%) e espessura da parede/diâmetro externo vascular (WT%). As expressões de TLR-4, MMP-9/TIMP-1 e PCNA em células de músculo liso vascular pulmonar foram detectadas, e suas relações com PVR foram então analisadas. RESULTADOS: As inflamações (1,6 ± 0,8), WA% (44,0 ± 6,4) e WT% (27,3 ± 3,3) no grupo COPD foram maiores que no grupo controle (0,3 ± 0,5; 26,1 ± 2,8; 15,6 ± 1,8). E as expressões de TLR-4 (31,4 ± 14,7) e MMP-9/TIMP-1 (2,2 ± 2,6) foram aumentadas em relação ao grupo controle (4,7 ± 4,5, 1,9 ± 1,2). Na análise de correlação, TLR-4 e MMP-9/TIMP-1 foram positivamente correlacionadas com as inflamações (r = 0,18; P <0,01), WA% (r = 0,68; P <0,01) e WT% (r = 0,73; P <0,01), bem como correlacionadas positivamente com a expressão de PCNA (r = 0,44; P <0,01). A elevação da TLR-4 foi correlacionada positivamente com as expressões de MMP-9 e TIMP-1. CONCLUSÕES: A regulação positiva do TLR-4 nas células do músculo liso arterial pulmonar de pacientes com DPOC poderia promover as inflamações e a expressão de MMP-9, causando assim uma degradação anormal da matriz extracelular, por isso desempenhou um papel importante no processo de PVR.

Expression of collagenases (matrix metalloproteinase-1, 8, 13) and tissue inhibitor of metalloproteinase-1 of retrodiscal tissue in temporomandibular joint disorder patients

OBJECTIVES: The aim of this study was to reveal how collagenases (matrix metalloproteinase [MMP]-1, 8, 13) and tissue inhibitor of metalloproteinase 1 (TIMP-1) are expressed in immunohistochemistry of retrodiscal tissue in temporomandibular joint disorder patients. MATERIALS AND METHODS: This study was conducted on 39 patients who underwent discoplasty or discectomy. Immunohistochemical staining was undertaken and expression levels of MMP-1, 8, 13, and TIMP-1 were evaluated. The status of internal derangement of disc, osteoarthritis, and joint effusion were analyzed using magnetic resonance imaging (MRI). Disc status observed during operation was also categorized. RESULTS: The more severe disc derangement was observed on MRI, the more increased expression of MMPs and TIMP-1 appeared. Regarding MMP-13 expression, 86.7% of late-stage disc displacement patients showed grade II or III. Expression level of MMPs or TIMP was not statistically significant associated with joint effusion level. In perforation and/or adhesion groups, all patients showed grade II or III expression of MMP-13. Once perforation occurred, MMP-13 showed increased expression with statistical significance. CONCLUSION: MMP-1 and MMP-13 expression seem to be related to progression of osteoarthritis whereas MMP-8 does not seem to have a specific role with regard to temporomandibular joint disorders. TIMP-1 is considered to be partly related to internal derangement rather than osteoarthritis, but it is not significant.

Salvianolic acid A attenuates ischemia reperfusion induced rat brain damage by protecting the blood brain barrier through MMP-9 inhibition and anti-inflammation / 中国天然药物

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.

Salvianolic acid A attenuates ischemia reperfusion induced rat brain damage by protecting the blood brain barrier through MMP-9 inhibition and anti-inflammation / 中国天然药物

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.