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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.
Subject(s)
Rats , Mice , Animals , Ureteral Obstruction/metabolism , Kidney/metabolism , Tissue Inhibitor of Metalloproteinase-1/metabolism , Molecular Docking Simulation , Rats, Sprague-Dawley , Kidney Diseases/drug therapy , Extracellular Matrix/metabolism , Flavonoids/metabolism , FibrosisABSTRACT
Tissue has a complex three-dimensional (3D) dynamic structure, and is affected by various forms of forces. Cells sense mechanical forces from extracellular matrix (ECM), and the mechanical micro-environment constructed by ECM regulates different biological functions of cells. To prepare biomaterials which can simulate the ECM mechanical micro-environment of tissues is one of the research hot spots and difficulties in biomechanical field. Different physical and chemical properties of biomaterials endow materials with specific mechanical properties, which further affect the behavior and function of cells. Based on the latest literature of biomechanics of materials in the year 2021, this study mainly focused on the role of novel mechanical biomaterials in regulating cell biological behavior and application in tissue engineering. The future development direction in the field of biomechanics of materials was also discussed.
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Objective To study the influence of cell-extracellular matrix (ECM) adhesion on migration of tumor cells regulated by ECM stiffness. Methods The cellular Potts model (CPM) was established to simulate tumor cell growth and cellular immune feedback system. The effects from mechanical behavior of cells on cell-ECM adhesion were observed, and the migration of tumor cells under different ECM was analyzed. Results The ECM stiffness could influence the migration rate of tumor cells. The change of ECM stiffness regulated the adhesion force between cells and ECM, and the change of adhesion force would influence the migration rate of cells. Conclusions The migration and distribution patterns of cells are closely related to the adhesion and stiffness of ECM. The increase in ECM stiffness can effectively promote the migration rate of tumor cells, and the further increase in ECM stiffness inhibits the migration of tumor cells. These findings may further reveal dynamic changes of ECM, adhesion and mechanical performance of tumor cell migration.
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Objective:To explore the effect of different effective parts of Taohe Chengqitang on the synthesis and degradation of extracellular matrix in human kideny-2（HK-2） cells induced by transforming growth factor-β1（TGF-β1）. Method:Petroleum ether extract, ethyl acetate extract, n-butanol extract, raffinate and polysaccharide extract, mirabilite extract were extracted with 70% ethanol by systematic solvent method. The HK-2 cell fibrosis model induced by TGF-β1 was built to intervene the cells in different parts of Taohe Chengqitang with different concentrations (0, 50, 100, 200, 400, 800 mg·L-1). Enzyme-linked immunosorbent assay（ELISA）kit assay was used to detect collagen（Col）-Ⅰα1 and fibronectin （FN）in supernatant to screen out the main active parts. Cell counting kit-8 （CCK-8）method was used to determine the best concentration of intervention site of bioactive components. Western blot analysis was used to detect the expression levels of Col-Ⅰ, Col-Ⅲ, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase inhibitor2 (TIMP2), and connective tissue growth factor (CTGF). Immunofluorescence assay was used to detect the expression of α-smooth muscle actin（α-SMA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） analysis was used to detect the mRNA expression of plasminogen activator inhibitor-1（PAI-1）. Result:ELISA kit assay demonstrated that compared with the model group, ethyl acetate extract, n-butanol extract and chloroform extract significantly reduced the Col-Ⅰα1 and FN content at the concentrations of 200 and 400 mg·L-1 (P<0.05, P<0.01). CCK-8 assay showed that the cells viability was significantly inhibited with drug intervention at the concentrations of 400 and 800 mg·L-1 (P<0.01). Western blot demonstrated that compared with the model group, ethyl acetate extract, n-butanol extract and chloroform extract decreased the expression levels of Col-Ⅰ, Col-Ⅲ, TIMP2 and CTGF in HK-2 cells induced by TGF-β1, and increased the expression of MMP-2 (P<0.05), with more significant effect in n-butanol extract (P<0.01). The results of immunofluorescence showed that ethyl acetate extract, n-butanol extract and chloroform extract could inhibit the expression of α-SMA (P<0.05), with more significant effect in n-butanol extract (P<0.01). The results of Real-time PCR showed that ethyl acetate extract and chloroform extract inhibited mRNA expression of PAI-1 (P<0.05), with more significant effect in n-butanol extract (P<0.01). Conclusion:The extracts of ethyl acetate, n-butanol and chloroform are the active parts of Taohe Chengqitang with the anti-renal fibrosis effect, with n-butanol extract as the most active part. The mechanism on anti-renal fibrosis may be related to its regulation of extracellular matrix (ECM) synthesis and degradation.
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Objective:To observe the effects of Fushengong prescription on secretory glycoprotein （Wnt）/β-serial protein （β-catenin） signaling pathway in kidney of rats with chronic renal failure （CRF），and to further explore its mechanism of releasing the aggregation of extracellular matrix（ECM），inhibiting renal tubule interstitial fibrosis （TIF） and prolonging the progression of CRF. Method:A total of 55 SD male rats were randomly divided into the normal group，the model group，and the low， medium and high dose groups of Fushengong prescription，with 11 rats in each group.The normal group was routinely reared and the other 4 groups of rats were used to establish CRF model with 0.5% adenine fodder， fed them continuously for 21 d. After successful modeling，all model rats were switched to conventional feed. Normal saline （NS） was given the normal group and the model group by 20 mL·kg-1·d-1， the low， middle and high dose groups rats of Fushengong prescription were given intragastric administration Fushengong prescription according to the body weight of 4， 8， 16 g·kg-1，once a day，continuous gavage for 30 d. After the experiment，the pathomorphism change of renal tissues of rats was measured by Masson staining， the expression of Wnt4 and β-catenin mRNA in the kidney tissues were observed by the method of Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， the expression of Wnt4，β-catenin and matrix metalloproteinase-7（MMP-7） protein of renal tissues were detected by the methods of Western blot. The expression of Wnt4， β-catenin protein of renal tissues were detected by the methods of immunohistochemistry （IHC）. Result:Compared with normal group，renal tubule interstitial fibrosis of renal tissues increased distinctly and the expression of Wnt4，β-catenin and MMP-7 protein increased significantly in the model group. Wnt4 and β-catenin mRNA also increased significantly in model group（P<0.01）. Compared with model group， the expression of Wnt4， β-catenin and MMP-7 protein in the Fushengong prescription groups decreased obviously （P<0.05）. The expression of Wnt4 and β-catenin mRNA in Fushengong prescription groups also decreased obviously. Conclusion:The mechanism of Fushengong prescription can release the aggregation of ECM，inhibit TIF and delay the progression of CRF，which may be related with the activation of Wnt/β-catenin signal pathway.
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Objective To investigate the influences of different matrix stiffness on proliferation ability and glucose metabolism of hepatocellular carcinoma (HCC) cells and to explore the correlation between metabolism and biological behavior changes of HCC cells resulted from the stiffness of extracellular matrix (ECM)．Methods The proliferation changes of HepG2 cells cultured on matrix with different stiffness were detected by CCK-8 assay and cell count assay. 2-NBDG and flow cytometry were used to detect the effect of matrix stiffness on glucose uptake. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of Glut1. Then, 2-DG was used to inhibit glycolysis, and the influences of matrix stiffness on proliferation of HepG2 cells were detected. Results The proliferation ability, glucose uptake and the expression of Glut1 of HepG2 cells increased with the matrix stiffness increasing. When glycolysis was inhibited, the proliferation ability of HepG2 cells grown on matrix with different stiffness was similar. Conclusions The mechanical microenvironment had an important effect on proliferation of HCC cells; matrix with a larger stiffness might promote proliferation of HCC cells through regulating glycolysis. The research findings provide a corresponding experimental basis for the clinical treatment of HCC cells and drug development targeting glucose metabolism.
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Objective: To investigate the effect of Kangxianling decoction on renal function and expression of extracellular matrix(ECM) in renal tissue of rats with renal fibrosis induced by 5/6 nephrectomy. Method: Totally 50 SD rats were randomly divided into control group (n=7), sham-operation group (n=7), operation group (n=36). The 5/6 nephrectomized renal fibrosis model was established in operation group. After successful modeling, rats were randomly divided into model group, Kangxianling group and losartan potassium group. The losartan potassium group and the Kangxianling group were given losartan potassium (33.3 g·kg-1) and Kangxianling decoction (21 g·kg-1) every day. The control group, sham-operation group and model group were all treated with equal volume of normal saline. Rats were put to death after 24 weeks of consecutive medication, and renal function, 24 h urine volume and 24 h-Pro quantitation of each group were measured. The expressions of collagen Ⅰ(ColⅠ) and ColⅢ in renal tissues were determined by immunohistochemistry and Western blot. Result: Compared with the normal group and sham-operation group, serum creatinine(SCr), blood urea nitrogen(BUN), 24 h urine volume, 24 h-Pro quantitation, renal tissue ColⅠ and ColⅢ expression levels were significantly increased in the model group (PPPConclusion: Kangxianling decoction can down-regulate the expressions of ColⅠ and ColⅢ in kidney tissue, there by inhibiting the accumulation of ECM, and exerting the effect in delaying renal fibrosis and protecting renal function.
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To evaluate the protective effects of glycosides/phenol component of Moutan Cortex (MC) on renal injury of diabetic nephropathy (DN) rats based on renal function parameters and histopathological examinations(HE staining and transmission electron microscope),and explore its possible mechanism by establishing DN rat models induced by high-sugar high-fat diet combined with streptozotocin (STZ). The results showed that compared with the model group, the MC glycosides/phenol component high and low dose groups(0.808, 0.404 g•kg⁻¹•d⁻¹) could significantly improve serum creatinine, blood urea nitrogen, urine protein and other abnormal renal function parameters. HE staining and transmission electron microscope results showed thickening of glomerular basement membrane, proliferation of mesangial cells and damages of podocyte structure in major rats of model group. However, the intervention of glycosides/phenol component of MC could effectively protect the glomerular injury. To explore its possible mechanism, the expressions of TGF-β1, fibronectin (FN) and collagen Ⅳ in renal tissues of rats in each group were detected by Western blot and immunohistochemical assay, and the phosphorylation levels of downstream effect factors (Smad2/3, p38MARK) of TGF-β1 were detected. The results showed that glycosides/phenol component of MC could effectively antagonize the activity of TGF-β1, lower the expressions of fibronectin (FN) and collagen Ⅳ inextracellular matrix (ECM), and resist against the thickening of glomerular basement membrane. More importantly, its protective effect on renal injury in DN rats may be associated with interfering the conduction of Smad, MARK pathways and resisting against the TGF-β1-induced ECM accumulation.
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Objective To provide an experimental basis for long-term in vitro proliferation,tissue construction and function maintaining of human primary hepatocytes by preparing three-dimensional (3D) porous specific extracellular matrix (ECM) derived from porcine liver with different decellularizing methods.Methods The porcine liver tissue slices were treated using six different decellularization/oxidation ways,and the decellularization extent and ECM structure were evaluated through qualitative and quantitative analysis.Results The decellularized liver-ECM could maintain the original shape during the whole process of the decellularization.HE staining showed that the cellular components and nucleus disappeared in the decellularized ECM,indicating that all of these six decellularization methods can completely decellularize the porcine liver tissue slices.Determination of DNA content showed that 91％ ～ 97％ of cell components have been removed.Electron microscopy scanning observed that the pore sizes and ultrastructures of the liver ECMs under six decellularization treatments were significantly different.Conclsion The six decellularization/oxidation methods evaluated in this study could successfully create a 3D specific decellularized liver ECM with porous ultrastructure,which further lays basis for the development of a novel in vitro 3D culture model for human hepatocytes.
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Objective To investigate the regulating effect and mechanism of microRNA-21 (miR-21) on extracellular matrix (ECM) of vascular smooth muscle cells (VSMCs) by vascular remodeling of hypertension. Methods By narrowing the abdominal aorta in rats, the hypertension models were established and divided into 2-week hypertension group and 4-week hypertension group, and sham-operated group was also established as control. VSMCs from the rat aorta were subjected to 0% (static), 5% (normal) and 15%（hypertensive）elongation strain at a constant frequency of 1.25 Hz and duration of 12 hours, respectively. The expressions of Smad 7 and ECM were detected by Western blotting, and the expression of miR-21 was examined by Real-time RT-PCR. Finally, miR-21 siRNA was used to study the role of miR-21 in the mechanical strain-induced expression of ECM, miR-21 and Smad 7. Results Compared with the sham-operated group, ECM and miR-21 in thoracic aorta of 2-week hypertension group were significantly elevated. Collagen I, collagen III and miR-21 in thoracic aorta of 4-week hypertension group were significantly elevated. Compared with the static and 5% strain groups, the protein expression of collagen I in VSMCs did not show significant change, but the protein expression of collagen III was significantly elevated and Smad 7 expression was significantly decreased in 15% strain group. The cyclic strain also enhanced miR-21 expression in VSMCs. miR-21 inhibitor effectively decreased the expression of miR-21 in VSMCs and protein level of collagen III, while enhanced Smad 7 expression under the static and 15% strain. Conclusions The vascular remodeling of hypertension causes the high expressions of ECM and miR-21. The cyclic strain induces the high expression of miR-21, which via Smad 7 results in enhancing the expression of ECM, collagen III especially, in VSMCs under vascular remodeling of hypertension.
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To analyze recent studies on the role of transient receptor potential vanilloid 4 (TRPV4) in fibrosis diseases and related mechanism. TRPV4 is a class of non-selective cation channel protein, which involved in intracellular divalent cations, mainly regulate Ca2+ homestasis and participate in the development of multiple organ fibrosis broadly. TRPV4 plays an important role in the prevention and treatment of fibrosis diseases. But for its complex mechanism of action, further research needs to be studied.
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Objective The aim of the study was to investigate the effects of inflammation on the biological be-haviors of the VF -MSCs and provide the theoretic basis for the repair of the vocal folds which were damaged by in-flammation .Methods The inflammatory vocal fold tissues and normal vocal fold tissues were respectively derived from the vocal cord polyp and normal tissues of the hypopharyngeal carcinoma patients .The HE staining ,masson trichrome staining and elastin van gieson (EVG) staining were performed to detect the effects of inflammation on the collagenous fiber and elastic fibers of the vocal fold lamina propria .The cell colony formation analysis and MTT cell growth curve were used to detect the effects of inflammation on the proliferation of VF -MSCs .The effects of in-flammation on the multi-directional differentiation of VF -MSCs were evaluated by inducing the VF -MSCs to dif-ferentiate into osteoblasts and lipoblasts .Results The results of masson trichrome staining and EVG staining showed that in the inflammatory vocal fold lamina propria collagen fibers became thicker and the amount of collagen fibers increased ,while elastic fibers became thinner and the amount of elastic fibers decreased .Compared with the vocal fold mesenchymal stem cell (VF-MSCs) in normal vocal folds ,VF-MSCs in inflammatory vocal folds pro-liferated more significantly ,but the osteogenic differentiation and adipogenic differentiation of VF -MSCs in inflammatory vocal folds were restrained .Conclusion Inflammation enhanced the compressive resistance ,abated the elasticity , and restrained the multi -directional differentiation of VF -MSCs .
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By preventing mesenchymal stem cells (MSCs) from adhering to precoated agarose to create a model of MSC suspension in vitro, we investigated anoikis in MSCs and the role of caspase-3 in the anoikis. The cultured MSCs were randomly divided into 3 groups: the anoikis group,caspase-3 inhibitor group and control group. Before experiment, we coated dishes with 1.5 % agarose;in the anoikis group, MSCs were put into the precoated dishes; and in the inhibitor group, caspase-3 inhibitor and MSCs were also put into the precoated dishes; but there were not intervention in the control group. MSCs were collected at 2 h, 6 h, 12 h and 24 h. The alteration of caspase-3 activity was evaluated by caspase-3 fluorometric assay and western blot analysis. The apoptosis rates were detected by flow cytometry. MSCs were round and suspended sufficiently in the anoikis and inhibitor groups. Caspase-3 fluorometric assay showed that there were significant differences in statistics between the anoikis group and the others (P＜0.05). Western blot analysis discovered that caspase-3 expression.in the anoikis group was more than that in the control and inhibitor groups (P＜0.05). Flow cytometry showed that the apoptosis peak appeared in all the three groups, but it increased dramatically in the anoikis group. The apoptosis rates in the inhibitor and control groups were low and stable.And there were significant differences in statistics between the anoikis group and the others (P＜0.05).MSCs will undergo anoikis in suspended condition if they are separated from the extracellular matrix.Caspase-3 inhibitors can suppress caspase-3 activity and reduce the apoptosis rate significantly. Caspase-3 plays a vital part in induced MSC anoikis in vitro. MSCs suspension culture system might be set up with argorose and caspase-3 inhibitor.
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Objective To study the effects of MMP-9 (Matrix Metalloproteinase-9, MMP-9) in the pathogenesis of abdominal aortic aneurysms (AAAs) by localizing the expression of MMP-9 in the aneurysmal tissues. Methods By means of immunohistochemistry, the frozen sections (5 μm) with aneurysmal tissues (n = 10) were incubated with MMP-9 antibody-added agents, then the sections were stained and observed under the microscope to localize the expression of MMP-9, which displayed a brown precipitate within the arterial walls. The normal arterial wall tissues(n= 10)and the diseased arterial wall tissues from the arterial occlusive diseases (AODs) (n= 15) were also immunized exactly the same way as control. Results A quantity of positive granules which appeared within the aortic media showed the strong expression of MMP-9 in the AAAs, with the positive rate reaching 95%(19/20), while no expression of MMP-9 was observed in the normal artery. However, the scattered distributed positive granules were scen within the arterial wall of some cases of the AODs, implying the weak positive expression of MMP-9 in this disease with the positive rate of 26.7%(4/15). There was a significant difference of the expression of MMP-9 within the arterial wall between the AAAs and AODs(P＜0. 01). Conclusion High expression of MMP-9 within the aortic media faciliatates the degradation of collagen and elastin fibres and subsequent dilation of the aortic artery , thus playing an important role in the pathogenesis of AAAs. To refrain MMP-9 from enhanced expressing within the aortic wall is of clinical significance in the prevention and treatment of AAAs.
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Objective:To investigate the effect of IRH on pulmonary fibrosis and its intervention mechanism.Methods:45 sprague-dawley rats were randomly divided into three groups.The model group and the intervention group were received endotracheal injection of bleomycln to induce pulmonary fibrosis,while the control group were received endotracheal injection of saline.From the second day the intervention group IRH were injected to the stomach,while the other two groups were injected saline instead.On the 7th day,14th day and 28th day the lungs were isolated.The expression of MMP-2 and TIMP-1 was detected by immunohistochemical method respectively.Results:There was only a very weak expression of MMP-2 and TIMP-1 in the control group,there was significantly higher level in model group compared with control group on the 7th day,14th day and 28th day.The IRH group was significantly lower than the model group with the same time points,though it was also obviously higher than the control group all over the period(P
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0.05).Urethroscopy showed a smooth and intact internal mucosa,wide urethral caliber and normal-appearing urethral tissue in the 2 groups. Conclusions VECM has the same regenerative process as UECM in the replacement of urethral defect.Moreover,VECM has wider source and better elasticity and mechanical properties,so VECM appears to be an ideal material for urethral replacement.
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Objective To investigate the effect of changes in plasma lipid levels on the accumulation of glomerular extracellular matrix in type 2 diabetic rats.Methods Rats were divided into three groups,namely,normal control,diabetes mellitus treated with Fenofibrate,which was gastrically administrated in the dose of 20mg?kg -1 ?d -1 for 22 weeks.A quantitative analysis of the components of glomerular extracellular matrix (ECM) were performed with immunoperoxide (ABC) method and the computer imagine analysis system.Results The result showed that diabetic rats had significantly higher plasma lipid levels and accumulation of ECM including collagen Ⅳ,laminine and fibronectin than the normal control ( P