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Objective·To explore the role of advanced platelet-rich fibrin(A-PRF)in osteochondral regeneration.Methods·Bone-marrow mesenchymal stem cells(BMSCs)and knee joint chondrocytes were obtained from New Zealand rabbits.A-PRF was obtained by low-speed centrifugation of the heart blood of rabbits.The histological structure of A-PRF was observed by an optical microscope.The release of growth factors in A-PRF was detected by ELISA,including platelet-derived growth factor,transforming growth factor-β,insulin-like growth factor,vascular endothelial growth factor,epidermal growth factor and fibroblast growth factor.A-PRF's cytotoxicity and capability for promoting the proliferation of rabbit BMSCs were detected by live/dead double staining and MTT methods.The effect of A-PRF on the gene expression of type Ⅱ collagen,aggrecan,alkaline phosphatase(ALP)and osteocalcin(OCN)in rabbit BMSCs was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Transwell chambers were used to determine the effect of A-PRF on the migration ability of rabbit BMSCs and the chondrocytes.Rabbit knee osteochondral defect models were established,and 18 rabbits were randomly divided into 3 groups.The A-PRF group(n=6)was implanted with A-PRF in the defect,the A-PRF+BMSCs group(n=6)was implanted with rabbit BMSCs on A-PRF,and the control group(n=6)did not undergo implantation.The rabbits were sacrificed 12 weeks after surgery and the knee joint specimens were stained with hematoxylin-eosin(H-E),toluidine blue and safranin O/fast green.Based on the surface morphology and histology of the knee joints,the International Cartilage Repair Society(ICRS)scoring system was used for macroscopic and histological scoring.Results·A-PRF had a loose network structure and can slowly release growth factors.No cytotoxicity to rabbit BMSCs was observed after adding A-PRF,and the the capability for promoting the proliferation of rabbit BMSCs was significantly increased at 24,48 and 72 h after adding A-PRF(all P<0.05).Chondrogenesis-related gene Ⅱ collagen and aggrecan,as well as osteogenesis-related genes ALP and OCN were significantly up-regulated(all P<0.05).After adding A-PRF,the migration abilities of rabbit BMSCs and chondrocytes were significantly enhanced(both P<0.05),and the migration ability of rabbit BMSCs was significantly higher than that of chondrocytes(P=0.025).The joint surface morphology in the rabbit knee joint defect models was observed.It can be seen that the defects in the A-PRF group and the A-PRF+BMSCs group were basically restored,while the the defects in the control group were only covered by soft tissue.In the ICRS macroscopic score,there was no statistical difference between the A-PRF group and the A-PRF+BMSCs group,but the scores of the two groups were all significantly higher than those of the control group(all P<0.05).According to the histological results,both the A-PRF group and the A-PRF+BMSCs group formed osteochondral repair,but the cartilage in the A-PRF group was more mature,while the control group formed fibrous repair.In the ICRS histological score,there was no statistical difference between the A-PRF group and the A-PRF+BMSCs group,but the scores of both the groups were significantly higher than those of the control group(both P<0.05).Conclusion·Autologous A-PRF has good biocompatibility and the capability for promoting the proliferation of BMSCs.It can promote the repair of cartilage and subchondral bone both in vitro and in vivo.
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ObjectiveTo investigate the role and mechanism of podoplanin （PDPN） in hepatic stellate cell （HSC） activation and liver fibrosis. MethodsLiver biopsy samples were collected from 75 patients with chronic hepatitis B who attended Department of Infectious Diseases， Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine， for the first time from September 2019 to June 2022， and RT-PCR and immunohistochemistry were used to measure the expression of PDPN in liver tissue of patients in different stages of liver fibrosis. A total of 12 male C57/BL6 mice were randomly divided into control group and model group. The mice in the model group were given intraperitoneal injection of 10% CCl4， and those in the control group were injected with an equal volume of olive oil， for 6 weeks. HE staining and Sirius Red staining were used to observe liver histopathological changes； primary mouse liver cells were separated to measure the mRNA expression of PDPN in various types of cells； primary mouse HSCs were treated with PDPN protein， followed by treatment with the NF-κB inhibitor BAY11-708， to measure the expression of inflammatory factors in HSCs induced by PDPN. The independent-samples t test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. The Spearman correlation analysis was used to investigate data correlation. ResultsAs for the liver biopsy samples， there was a relatively low mRNA expression level of PDPN in normal liver， and there was a significant increase in the mRNA expression level of PDPN in liver tissue of stage S3 or S4 fibrosis （all P<0.001）. Immunohistochemical staining showed that PDPN was mainly expressed in the fibrous septum and the hepatic sinusoid， and the PDPN-positive area in S4 liver tissue was significantly higher than that in S0 liver tissue （t=8.892， P=0.001）. In normal mice， PDPN was mainly expressed in the hepatic sinusoid， and there was a significant increase in the expression of PDPN in CCl4 model mice （t=0.95， P<0.001）， mainly in the fibrous septum. RT-PCR showed a significant increase in the mRNA expression of PDPN in the CCl4 model mice （t=11.25， P=0.002）. Compared with hepatocytes， HSCs， Kupffer cells， and bile duct endothelial cells， hepatic sinusoidal endothelial cells showed a significantly high expression level of PDPN （F=20.56， P<0.001）. Compared with the control group， the primary mouse HSCs treated by PDPN protein for 15 minutes showed significant increases in the mRNA expression levels of the inflammation-related factors TNFα， CCL3， CXCL1， and CXCR1 （all P<0.05）， and there were significant reductions in the levels of these indicators after treatment with BAY11-7082 （all P<0.05）. ConclusionThere is an increase in the expression of PDPN mainly in hepatic sinusoidal endothelial cells during liver fibrosis， and PDPN regulates HSC activation and promotes the progression of liver fibrosis via the NF-κB signaling pathway.
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ObjectiveTo investigate the mechanism of action of Xiayuxue decoction in inhibiting nonalcoholic fatty liver disease （NAFLD） induced by high-fat diet in mice by regulating nucleotide binding oligomerization domain like receptor containing pyrin domain protein 6 （NLRP6）. MethodsA total of 15 male C57BL/6 mice were randomly divided into low-fat diet （LFD） group， high-fat diet （HFD） group， and Xiayuxue decoction-HFD group （XYXD group）， with 5 mice in each group. Liver function parameters （alanine aminotransferase ［ALT］ and aspartate aminotransferase ［AST］） and blood lipid metabolic indicators （triglycerides ［TG］ and total cholesterol ［TC］） were measured； HE staining and oil red O staining were performed for liver tissue to observe histomorpholoty and lipid droplet deposition； quantitative real-time PCR was used to measure the expression levels of inflammatory factors （tumor necrosis factor-α ［TNF-α］， interleukin-1β ［IL-1β］， interleukin-18 ［IL-18］， and NLRP6） in liver tissue； Western blot was used to measure the protein expression levels of NLRP6， nuclear factor-kappa B （NF-κB）， and NF-κB p65； immunohistochemistry was used to measure the expression of NLRP6 and CD68. Mouse Raw264.7 cells were treated with palmitic acid （PA）， lipopolysaccharide， and serum containing Xiayuxue decoction to observe inflammation. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the LFD group， the HFD group had significant increases in the serum levels of ALT， AST， TC， and TG （all P<0.05）. Liver histopathological examination showed that the HFD group had marked hepatic steatosis and a signficant increase in NAS score （P<0.05）， and quantitative real-time PCR showed significant increases in the inflammatory factors such as IL1β and IL-18 and a significant reduction in the expression of NLRP6 （all P<0.05）. Immunohistochemistry showed that the expression of NLRP6 showed a similar trend as that of the macrophage marker CD68. Western blot showed that after the downregulation of NLRP6 expression， there was a significant increase in phosphorylated NF-κB p65 （P<0.05）. Compared with the HFD group， Xiayuxue decoction effectively improved liver inflammation， upregulated the expression of NLRP6， and downregulated phosphorylated NF-κB p65 in HFD mice （all P<0.05）. After Raw264.7 cells were treated with PA， NLRP6 was downregulated to promote the progression of inflammation （P<0.05）， and treatment with Xiayuxue decoction could upregulate NLRP6 and inhibit inflammation NF-κB （P<0.05）. ConclusionXiayuxue decoction can effectively improve hepatic steatosis and liver inflammation in a mouse model of NAFLD， possibly by regulating NLRP6/NF-κB to alleviate macrophage activation.
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Objective To investigate the therapeutic effect of Taohong Siwu decoction on a mouse model of carbon tetrachloride (CCl 4 )-induced liver fibrosis and its mechanism of action. Methods A total of 24 male C57BL/6 mice were randomly divided into normal group, model group, and Taohong Siwu decoction group, with 8 mice in each group. The mice in the model group and the Taohong Siwu decoction group were given intraperitoneal injection of 10% CCl 4 , and Taohong Siwu decoction was given by gavage since week 3 for 4 consecutive weeks. Liver function [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] was measured, and liver pathomorphology was observed. Real-time PCR was used to measure the mRNA expression of α-smooth muscle actin (α-SMA), hyaluronic acid synthase-2 (HAS-2), and collagen type Ⅰ(Col1), and Western blotting was used to measure the protein expression of α-SMA, Col1, and HAS-2. Primary hepatic stellate cells (HSCs) were isolated, and HAS-2 was silenced by siRNA to observe its influence on HSC activation. The t -test was used for comparison of continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the SNK or least significant difference t -test was used for further comparison between two groups. Results Compared with the normal group, the model group had significant increases in serum liver function parameters (ALT, AST) and the Taohong Siwu decoction group had significant reductions in the serum levels of ALT and AST (all P < 0.01). Pathological staining showed that the model group had marked inflammatory cell infiltration and formation of fibrous septa by proliferated collagen fibers, and the Taohong Siwu decoction group had loose fibrous septa and alleviated inflammatory cell infiltration. Compared with the model group, the Taohong Siwu decoction group had significant reductions in the mRNA and protein expression of α-SMA and Col1(all P < 0.001). Compared with the normal group, the model group had a significant increase in the mRNA expression level of HAS-2 in liver tissue ( t =6.14, P < 0.05), and compared with the model group, the Taohong Siwu decoction group had a significant reduction in the protein expression level of HAS-2 (0.29±0.10 vs 1.00±0.12, t =70.73, P < 0.001). After HAS-2 was silenced by siRNA, the Si HAS-2+transforming growth factor β (TGFβ) group (treated with TGFβ) had significant reductions in the mRNA expression levels of α-SMA and Col-Ⅰ compared with the NC+TGFβ group ( P < 0.01). Conclusion Taohong Siwu decoction exerts a marked therapeutic effect on CCl 4 -induced liver fibrosis in mice by inhibiting HAS-2.
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In many pathogens infection,especially virus,antibody-dependent enhancement(ADE) can aggravate the infection and lead to severe diseases.In this immunopathological phenomenon,virus-specific antibodies enhance the entry of virus into monocytes,macrophages and granulocytic cells and even the replication of virus through different mechanism.This phenomenon has been reported in numerous pathogens including virus,bacteria and parasite and the mechanisms of ADE vary from different species.Further study of ADE can promote the vaccine research and development to make the most use of vaccine and prevent human body from pathogens,which will be helpful to control the spread of pathogens including Zika virus.In the present review,we review the research progress of ADE mechanism in recent years,including antibodies mediating,receptors mediating,complement mediating,viral proteins mediating and cellular mediating ADE.In addition,dengue virus,human immunodeficiency virus,Coxsackie virus,Ebola virus,Zika virus and other pathogens will be illustrated respectively.This review provides insights on the different mechanism of ADE in different pathogens.
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Objective To establish mouse models of intracerebral hemorrhage using autologous arterial blood,to study the physiological property of hippocampal neurons,brain edema changes and learning ability in the mouse models after intracerebral hemorrhage.Methods Eighty male C57/BL6 mice were randomly divided into intracerebral hemorrhage group and control group (n=40); 20 μL arterial blood from the tail arteries or normal saline were injected into the caudate nucleus of intracerebral hemorrhage group and control group by stereotactic technique,respectively.One,three,five and seven d after injection,the neurological impairment was scored; the behavioral changes of the mice in the Morris water maze (navigation test and space exploration experiment) were observed; brain edema was measured by wet and dry weight method and electrophysiological differences of hippocampal neurons were recorded by whole-cell patch-clamp technique and computer software.Results As compared with those in the control group,significantly increased neurological deficit scores one,three,five and seven d after injection,statistically decreased residence time in the platform on the fifth d of training,obviously increased water content around the brain edema one,three,five and seven d after injection,and significantly decreased resting membrane potential and input resistance in the hippocampal CA1 pyramidal cells five d after injection of mice in the intracerebral hemorrhage group were noted (P＜0.05).Conclusion The hippocampus-dependent spatial leaming ability of intracerebral hemorrhage mice is decreased,and the permeability of potassium channels is enhanced.
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Objectives To study the effect of cooling blood and invigorating circulation method on prevention and treatment of pulmonary fibrosis,and explore its mechanism.Methods Male ICR mice were randomly divided into 4 groups:negative control group,pulmonary fibrosis model group,cooling blood and invigorating circulation group and prednisone positive control group.The pulmonary fibrosis model of mice were established by BLM nasal instillation.After 8 hours,the mice were given prescription of cooling blood and invigorating circulation(Xijiao Dihuang decoction) by gastric perfusion.At the 28th day after treatment,the indexes were detected.The TGF-?1 content of bronchoalveolar lavage fluid(BALF) was tested by enzyme linked immunosorbent assay,and hydroxyproline(HYP) was examined by alkaline hydrolysis.Results The degree of airsacculitis and fibrosis in model group are obviously higher than the negative control group by pathological observation.Compared with model group,HYP content was decreased obviously in herb group(P