Effects of drug serum of anti-fibrosis I herbal compound on calcium in hepatic stellate cell and its molecular mechanism

AIM: To investigate the effects of anti-fibrosis I herbal compound on intracellular Ca2+ in activated hepatic stellate cell (HSC) and to try to survey its molecular mechanism in treatment and prevention of hepatic fibrosis and portal hypertension. METHODS: The activated HSC line was plated on small glass cover slips in 24 wells culture dishes at a density of 5×106 /mL, and incubated in RPMI-1640 media for 24 h. After the cells were loaded with Fluo-3/AM, intracellular Ca2+ was measured with laser scanning confocal microscopy (LSCM). The dynamic changes of intracellular Ca2+, stimulated by carbon tetrachloride, TGF-β1 antibody and the drug serum of anti-fibrosis I herbal compound and under orthogonal design were determined by LSCM. The effect of anti-fibrosis I herbal compound on intracellular Ca2+ was observed before and after the addition of TGF-β1 antibody. RESULTS: The intracellular Ca2+ were significantly different in different dosage of carbon tetrachloride anti-fibrosis I formula drug serum, TGF-β1 antibody and different turn of these substance, but their interval time between CCl4 and TGF-β1 antibody, CCl4 and anti-fibrosis I drug serum had no influence on intracellular Ca2+. The result showed intracellular Ca2+ wasn't significantly different between rat serum without anti-fibrosis I and untreated group. After carbon tetrachloride stimulation, intracellular Ca2+ of activated HSC increased significantly when the dosage of CCl4 from 5 to 15 mmol/L, however, decreased significantly after stimulation by 5-20 μg/mL TGF-β1 antibody or 5-20 mL/L drug serum. Moreover, before and after the addition of TGF-β1 antibody, intracellular Ca2+ was significantly different. These results suggested that the molecular mechanism was independent of blocking TGF-β1 effects. CONCLUSION: Anti-fibrosis I herbal compound may treat hepatic fibrosis and decrease portal hypertension by inhibiting activated HSC contractility through decrease of intracellular Ca2+.

Anti-inflammation and anti-fibrosis actions of resveratrol on experimental pneumoconiosis

OBJECTIVE To investigate the therapeutic effects and related signaling pathways involved in the actions of resveratrol on experimental pneumoconiosis in vivo and in vitro.METHODS The pneumoconiosis animal model was induced by exposing male SD rats to 15mg·m-3 silica aerosol in an inhalation chamber system for 6h·d-1,5d·week-1 for up to 8 weeks.The vehicle or resveratrol(10or 20mg·kg-1)was preventively or remedially administered to the rats during or after the 4-or 8-week silica exposure(SE)period,respectively.After 4-,8-,and up to 14-week treatment,in vivo near-infrared fluorescence imaging analysis and histological analysis were performed to evaluate the pathological changes in rat lung.Inflammatory cytokines level in bronchoalveolar lavage fluid(BALF)was measured by ELISA testing,and the deposition of fibrotic collagen proteins in lung parenchyma was determined by western blotting and immunohistochemistry analysis.Microarray analysis was performed to screen the signaling pathways involved in the actions of resveratrol on pneumoconiosis in vitro models.Anti-inflammation action and signaling of resveratrol was evaluated on silica-stimulated rat alveolar macrophage,which is one of the crucial effector cells for silica-induced inflammatory response;anti-fibrosis action and signaling of resveratrol was evaluated on TGF-β-induced human lung fibroblast,which acts as a promoter in the later fibrotic process of pneumoconiosis.RESULTS Silica aerosol exposure significantly increased macrophage infiltration and matrix metalloproteinases activity in lung tissue concomitant with the increased levels of inflammatory mediators in BALF.Preventive treatment with resveratrol(20mg·kg-1·d-1)reversed all these biochemical indices as well as histopathological alterations induced by silica exposure.Post-SE resveratrol treatment mildly reduced silica-induced inflammatory response in rat lung with no statistical significance.In vitro study revealed that resveratrol could inhibit alveolar macrophage cell death and decrease the levels of IL-1β and TNF-αinduced by silica particle exposure to cultured alveolar macrophages.Resveratrol was further shown to inhibit the nuclear transition of NF-κB and formation of cleaved caspase-1.Encouragingly,resveratrol preventively attenuated the lung fibrosis,evidenced by less fibrotic nodules formation and collagen proteins expression.No significant improvement on lung fibrosis was observed with post-SE resveratrol treatment.Invitrostudy further demonstrated that resveratrol suppressed TGF-β-induced lung fibroblast proliferation and collagen deposition,concomitant with the depressed activity of TGF-β/Smad signaling in lung fibroblast.CONCLUSION Resveratrol shows the anti-inflammation and anti-fibrosis actions on experimental pneumoconiosis in vivo and in vitro models.The depression of NF-κB,NALP3-inflammasome,and TGF-β/Smad signaling pathways may be involved in the anti-inflammation and anti-fibrosis actions of resveratrol,respectively.Resveratrol could be a potential therapeutic agent for the intervention of pneumoconiosis.

An Ac-SDKP analogue resistant to angiotensin converting enzyme exhibits anti-fibrosis effects and improves heart function in mice after myocardial infarction

To explore the effect of an Ac-SDKP analog on left ventricular remodeling after myocardial infarction,we synthesized the analog Ac-SD_DK_DP by replacing Asp and Lys with their D isomers.The biological activities of Ac-SD_DK_DP were confirmed using flow cytometry,qRT-PCR,Western blots and fluorescence microscopy.The protective effects of Ac-SD_DK_DP on infarcted hearts were assessed in mice with myocardial infarction（MI）.The half-life of Ac-SD_DK_DP was prolonged to over 2 h from a few minutes that Ac-SDKP has.Compared with Ac-SDKP,the analog exhibited stronger inhibition on the differentiation of macrophages,expression of arginase I（ARG I） and TGF-β1 in mature macrophages,proliferation and secretion of collagen type I in cardiac fibroblasts.In MI mice mode,Ac-SD_DK_DP decreased collagen deposition and TGF-β1 expression in myocardium,thus improving the FS（%） to 23.0±7.8 compared with 11.2±6.2 in untreated mice and 11.7±5.3 in Ac-SDKP treated mice（P0.05）.This work shows that the Ac-SDKP analogue is potentially useful for protective treatment for heart failure post-MI.In addition,the anti-fibrosis mechanism of Ac-SDKP was correlated with the alternative activation（M2） of macrophages by assessing ARG I and TGF-β1,two important fibrosis-related molecules secreted in M2 macrophages.

Synthesis, anti-fibrosis activity, and quantitative structure-activity relationship studies of 1,3-disubstituted-pyridin-4(1H)-one derivatives

A series of 1,3-disubstituted-pyridin-4（1H）-one derivatives were synthesized. The results of a viability assay on NIH_T3 cells indicated that compound 3m potently inhibited the cell viability with an IC50 value of 2.0 μM. The 3D-quantitative structure-activity relationship analyses of 30 final molecules applying topomer CoMFA and AutoGPA methods gave two reasonable models with a cross-validated correlation coefficient q^2 of 0.662 and 0.787, respectively. The achievement herein suggested the application of 3-hydroxypyridin-4（1H）-one as a novel scaffold for the discovery of anti-fibrosis agents. In addition, the QSAR and pharmacophore models established with the activity data may provide new insights into the structure optimization of pyridin-4（1H）-one derivative with potent anti-fibrotic effects.

Exosome-mediated aptamer S58 reduces fibrosis in a rat glaucoma filtration surgery model

AIM:To confirm whether exosome-mediated delivery of aptamer S58(Exo-S58) has a better antifibrotic effect than naked S58 in human conjunctival fibroblasts(HCon Fs) and a rat glaucoma filtration surgery(GFS) model.METHODS:To enhance the effective reaction time of aptamer S58 in vivo, we loaded aptamer S58 into exosomes derived from HEK293 T cells by PEI transfection to determine the effect of Exo-S58 in HCon Fs and a rat GFS model.RESULTS:Exo-S58 can significantly reduce cell proliferation, migration and fibrosis in TGF-β2-induced HCon Fs. In an in vivo experiment, Exo-S58 treatment prolonged filtering bleb retention and reduced fibrosis compared with naked S58 treatment in GFS rats.CONCLUSION:The exosomes are safe and valid carriers to deliver aptamers. Furthermore, Exo-S58 exhibited superior antifibrotic effect than naked S58 both in HCon Fs cells and rat GFS models.

Progress in drug delivery system for fibrosis therapy

Fibrosis is a necessary process in the progression of chronic disease to cirrhosis or even cancer,which is a serious disease threatening human health.Recent studies have shown that the early treatment of fibrosis is turning point and particularly important.Therefore,how to reverse fibrosis has become the focus and research hotspot in recent years.So far,the considerable progress has been made in the development of effective anti-fibrosis drugs and targeted drug delivery.Moreover,the existing research results will lay the foundation for more breakthrough delivery systems to achieve better anti-fibrosis effects.Herein,this review summaries anti-fibrosis delivery systems focused on three major organ fibrotic diseases such as liver,pulmonary,and renal fibrosis accompanied by the elaboration of relevant pathological mechanisms,which will provide inspiration and guidance for the design of fibrosis drugs and therapeutic systems in the future.

woLiver Regeneration Effect of Oncostatin M Following Hepatectomy for the Rat Cirrhotic Liver Model

Background/Aims: Liver resection represents the treatment of choice for hepatocellular carcinoma (HCC) arising in well-compensated cirrhosis. Gene expression of the multifunctional cytokine, Oncostatin M (OSM), stimulates liver regeneration and adenoviral vector expressing OSM (AdOSM) allows a persistent expression of the gene. The aim of this study is to evaluate the benefits of the preoperative injection of AdOSM to the remnant lobes to regenerate the liver. Methods: A 70% partial hepatectomy was performed in dimethylnitrosamine-administrated cirrhotic rats with a preoperative injection of AdOSM, adenoviral vector carrying β-galactosidase (AdLacZ), or phosphate-buffered saline (PBS). The morphologic, histologic, and biochemical changes in the remnant liver and survival rates were then assessed. Results: Portal injection with clamping the portal branches of the resected lobes for 5 min made it possible to effectively transduce the adenoviral vector into the remnant lobes. The ratio of the remnant liver weight/body weight (%) was 2.3 ± 0.5 in the AdOSM group, 1.1 ± 0.3 in the AdLacZ group (p < 0.001), and 1.6 ± 0.4 in the PBS group (p = 0.02). The fibrous ratio (%) was 21.3 ± 4.6 in the AdOSM group and 35.2 ± 4.5 in the AdLacZ group on day 4 after hepatectomy and fibrous status was significantly decreased in the AdOSM group (p = 0.02). Serum hyaluronic acid which is the indicator of liver fibrosis was 215 ± 141 ng/mL in the AdOSM group and 1963 ± 1225 ng/mL in the AdLacZ group (p = 0.03). Conclusions: The OSM gene therapy may increase the possibility of hepatectomy in a cirrhotic liver by improving fibrosis, hepatic function, and hepatocyte regeneration.

The pharmacological mechanism of quercetin as adjuvant therapy of COVID-19

The emergence of novel severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)has caused the global outbreak and major public health concern.After the outbreak human-to-human transmission was confirmed with or without symptoms of upper and lower respiratory tract involvement.Up to date,there has been evidence that COVID-19 is beyond that of a typi­cal pulmonary disease and revealing pathomechanics of COVID-19-associated acute respiratory distress syndrome(CARDS),which include severe inflammation and pulmonary edema leading to impaired alveolar homeostasis,and resulting in an alter­ation of lung physiology,lung fibrosis,inflammation of endothelium,vascular thrombosis,as well as exaggerated immune re­sponse.Concerning this pathophysiology,the use of quercetin as phytotherapeutic may merit in the management of COVID-19 patients.In this review,the authors wish to elaborate on the molecular effect of quercetin on SARS-CoV-2 by giving a detailed mechanism of quercetin against the binding of the S-protein of the virus to angiotensin-converting enzyme 2(ACE2)recep­tors,the main protease(M^(pro))or 3C-like protease(3CL^(pro)),papain-like protease(PL^(pro)),and RNA-dependent RNA polymerase(RdRP).Recent clinical evidence supporting the use of quercetin in COVID-19 management is also discussed in this paper.

Direct intercellular communications dominate the interaction between adipose-derived MSCs and myofibroblasts aqainst cardiac fibrosis

The onset of cardiac fibrosis post myocardial infarction greatly impairs the function of heart. Recent advances of cell transplantation showed great benefits to restore myocardial function, among which the mesenchymal stem cells （MSCs） has gained much attention. However, the underlying cellular mechanisms of MSC therapy are still not fully understood. Although paracrine effects of MSCs on residual cardiomyocytes have been discussed, the amelioration of fibrosis was rarely studied as the hostile environment cannot support the survival of most cell populations and impairs the diffusion of soluble factors. Here in order to decipher the potential mecha- nism of MSC therapy for cardiac fibrosis, we investi- gated the interplay between MSCs and cardiac myofibroblasts （mFBs） using interactive co-culture method, with comparison to paracrine approaches, namely treatment by MSC conditioned medium and gap co-culture method. Various fibrotic features of mFBs were analyzed and the most prominent anti-fibrosis effects were always obtained using direct co-culture that allowed cell-to-cell contacts. Hepatocyte growth factor （HGF）, a well-known anti-fibrosis factor, was demonstrated to be a major contributor for MSCs＇ anti-fibrosisfunction. Moreover, physical contacts and tube-like structures between MSCs and mFBs were observed by live cell imaging and TEM which demonstrate the direct cellular interactions.

Adipose-derived stem cells inhibit dermal fibroblast growth and induce apoptosis in keloids through the arachidonic acid-derived cyclooxygenase-2/prostaglandin E2 cascade by paracrine

Background:The clinical features of keloids consist of aberrant proliferation,secretion,differentiation and apoptosis of keloid dermis-derived fibroblasts(KFBs).Notably,the apoptosis rate of KFBs is lower than the proliferation rate.Though the anti-fibrotic effect of adipose-derived stem cells(ADSCs)on keloids has become a hot topic of research,the exact anti-fibrotic mechanism of the paracrine effect remains unclear.This study aimed to find out how the conditioned medium of ADSCs(ADSC-CM)exerts an anti-fibrotic effect in KFBs.Methods:KFBs and ADSCs were extracted and cultured.Then,ADSC-CM was prepared.Whether ADSC-CM could inhibit KFB growth and induce apoptosis was verified by the use of a cell counting kit-8,an 5-Ethynyl-2-deoxyuridine(Edu)kit and flow cytometry.The expressions of cyclooxygenase-1(COX-1),COX-2,caspase 3 and B-cell lymphoma-2(Bcl-2)in ADSC-CM-cultured KFBs were tested by real-time PCR and western blotting.To clarify the role of COX-2 in ADSC-CM-induced KFB apoptosis,a specific COX-2 inhibitor,celecoxib,was applied to KFBs cultured in ADSC-CM.Moreover,we tested the production of arachidonic acid(AA)and prostaglandin E2(PGE2)by ELISA.Then,we established a keloid transplantation model in a nude mouse to validate the therapeutic effect in vivo.Results:The proliferation ability of KFBs cultured in ADSC-CM was found to be weakened and apoptosis was significantly increased.Caspase 3 expression was significantly upregulated and Bcl-2 was downregulated in ADSC-CM-cultured KFBs.Furthermore,ADSC-CM strikingly elevated COX-2 mRNA and protein expressions,but COX-1 expression was unaltered.COX-2 inhibitors reduced ADSC-CM-induced apoptosis.Additionally,COX-2 inhibition blocked the elevation of caspase 3 and reversed the decrease in Bcl-2 expression.ADSC-CM increased PGE2 levels by 1.5-fold and this effect was restrained by COX-2 inhibition.In the nude mouse model,expressions of AA,COX-2 and PGE2 were higher in the translated keloid tissues after ADSC-CM injection than in the controls.Conclusions:We showed activation of the COX-2/PGE2 cascade in KFBs in response to ADSC-CM.By employing a specific COX-2 inhibitor,COX-2/PGE2 cascade activation played a crucial role in mediating the ADSC-CM-induced KFB apoptosis and anti-proliferation effects.