Qiteng Xiaozhuo granules(芪藤消浊颗粒)medicated serum inhibits excessive proliferation and promotes apoptosis of human glomerular mesangial cells by targeting fat mass and obesity associated proteins

OBJECTIVE:To explore whether fat mass and obesity associated proteins(FTO) is an important target of Qiteng Xiaozhuo granules(QTXZG,芪藤消浊颗粒) medicated serum in regulating proliferation and apoptosis of glomerular mesangial cells.METHODS:Medicated serum was obtained from Sprague-Dawley(SD) rats administered intragastrically with QTXZG decoction.The optimal concentration and intervention time of medicated serum were selected with the cell counting kit 8 assay.Cell proliferation was assessed by 5-ethynyl-2’-deoxyuridine(Ed U) and cell apoptosis was investigated using flow cytometry.The expression of FTO,Proliferating cell nuclear antigen,Cyclin D1,B-cell lymphoma 2(Bcl2) and BCL2 assaciated X was detected by Western blot and Real-time quantitative polymerase chain reaction,respectively.Quantification of the m6A RNA methylation was utilized to determine the total level of m6A methylation modification.RESULTS:Ed U and flow cytometry assays revealed that QTXZG medicated serum can remarkably inhibit proliferation and promote apoptosis of lipopolysaccharide(LPS)-induced human glomerular mesangial cells(HGMCs).The FTO overexpression plasmid could inhibit proliferation and promote apoptosis of LPS-induced HGMCs.The FTO inhibitor(FB23-2) can significantly attenuate the effect of QTZXG medicated serum on inhibiting excessive proliferation and promoting apoptosis.QTXZG medicated serum can significantly increase FTO expression and decrease the level of m6A methylation modification.CONCLUSIONS:FTO is a key target for QTXZG medicated serum in inhibiting excessive proliferation and promoting apoptosis of human glomerular mesangial cells.

Bushen Qiangji Granule (补肾强脊颗粒) Medicated Serum Inhibits Osteogenic Differentiation of Fibroblasts in AnkylosingSpondylitis by Inhibiting the BMP/Smads Signal Pathway in vitro

Objective: To explore the mechanism of Bushen Qiangji Granule(补肾强脊颗粒, BSQJ) in restraining the osteogenic differentiation of ankylosing spondylitis(AS) fibroblasts. Methods: Hip joint capsules were obtained from AS patients(n=10) receiving total hip replacement and healthy hip joint capsules from patients with hip fracture(n=10) receiving surgery as a control. Finite fibroblast lines were established from these tissue samples to observe the effect of BSQJ on suppressing osteogenic differentiation of fibroblasts. The expression of osteogenic marker gene corebinding factor a1(Cbfa1) and Smad family proteins were examined by Western blot and real-time quantitative polymerase chain reaction(q PCR). Results: The m RNA expression level of Cbfa1 was significantly higher in AS fibroblasts than that in normal fibroblasts and the expression of p Smad1, p Smad5, Smad4 and Cbfa1 in AS fibroblasts was also higher, demonstrating the activation of the BMP/Smads signal pathway in AS fibroblasts. BSQJ-medicated serum not only restrained the m RNA and protein expression levels of Cbfa1 and inhibited protein expression level of Smad4 but also decreased the expression quantities of p Smad1 and p Smad5. Conclusions: BSQJ can inhibit osteogenic differentiation of AS fibroblasts in vitro by suppressing the activation of the BMP/Smads signal pathway. This may be the important molecular mechanism of BSQJ in regulating AS ossification.

Gengnianchun recipe inhibits apoptosis of pheochromocytoma cells from beta-amyloid 25-35 insult, better than monotherapies and their compounds

This study aims to determine and compare the protective effects of Gengnianchun recipe drug serum and compounds of its representative drug monotherapies against sympathetic nerve pheochromocytoma cell line PC12 cells damaged by beta-amyloid 25-35 at the cellular apoptosis and related signal pathway levels. PC12 cells cultured with medicated rat serum showed enhanced cell viability and reduced cellular apoptosis rates compared with those of monotherapies and their compounds. Furthermore, Gengnianchun recipe up-regulated expressions of anti-apoptotic protein Bcl-2, estrogen receptor-beta and phosphorylated extracellular-signal-regulated kinase 1/2; and down-regulated expressions of pro-apoptotic proteins Bax and caspase-3. Gengnianchun recipe was superior to representative drug monotherapies, such as paeoniflorin, berberine, timosaponin A-III, icariine and their compounds in protecting PC12 cells. Mitogen-activated protein kinase blocker and estrogen receptor antagonist were found to reverse the above effects of Gengnianchun recipe. The experimental findings indicate that, Gengnianchun recipe protects PC12 cells from beta-amyloid 25-35 insult; its inhibitory effect on apoptosis may be achieved through the mitogen-activated protein kinase and estrogen receptor pathways.

Effect of Qishen decoction on dedifferentiation of sinusoidal endothelial cells by autophagy

Objective:To observe the effect of Qishen decoction on dedifferentiation and autophagy of liver sinusoid endothelial cells(LSEC);Methods:LSEC were randomly divided into the control group,model group,Qishen Decoction low,medium,high dose group,and inhibitor group.The model was induced by 100μg/ml oxidized low-density lipoprotein(oxLDL)for 24 hours,and the corresponding drugs or medicated serum were given for intervention.The expression levels of VEGFR2 and ET1 were detected by RT-qPCR and immunofluorescence staining,the ultrastructure of LSEC was detected by transmission electron microscopy,the content of NO was detected by ELISA,the expression levels of autophagy related proteins(LC3BI,LC3BⅡand p62)and endothelial function related proteins(eNOS and p-eNOS)were detected by western blot;Results:The results of transmission electron microscopy showed that Qishen decoction medicated serum could increase the number of fenestra and autophagy in LSEC cells,and inhibit the formation of basement membrane under endothelium.Compared with the model group,Qishen decoction medicated serum could significantly up-regulate the expression level of VEGFR2 mRNA and protein in LSEC,down regulate the expression level of ET1 mRNA and protein,the difference was statistically significant(P<0.05).In addition,Qishen decoction medicated serum could significantly increase the expression of LC3BII,p-eNOS,eNOS protein and the ratio of LC3BII/LC3BI,p-eNOS/eNOS,and reduce the expression of LC3BI and p62 protein in LSEC,which is statistically significant compared with the model group(P<0.05).Conclusion:Qishen decoction can inhibit the dedifferentiation of LSEC by promoting the autophagy level of LSEC,and then play an anti-fibrosis role.

Effects of Qingguang'an(青光安)containing serum on the expression levels of autophagy-related genes in human Tenon's fibroblasts induced by transforming growth factor beta 1

OBJECTIVE:To explore the effects of Qingguang'an(青光安)containing serum on the expression levels of autophagy related genes in the transforming growth factor beta 1(TGF-β1)-activated human Tenon's fibroblasts(HTFs).METHODS:(a)Primary HTFs were stimulated by TGF-β1 and underwent immunohistochemistry,which established a cell model after Glaucoma filtration surgery(GFS).(b)The cell models were divided into 4 group:normal group(normal cells),model group(+TGF-β1),treatment group(+TGF-β1+medicated serum),and positive control group(TGF-β1+rapamycin).Then,Qingguang'an medicated serum with optimum concentration was added to the corresponding group.The autophagy positive cells were identified by the Cyto-ID autophagy detection kits under fluorescent microscope and Cytation 5 multifunctional instrument for cell imaging.And the mean fluorescence intensity of autophagy positive cells was determined by flow cytometry.The expression levels of autophagy related genes—Beclin-1,autophagy related gene 5(ATG-5),and microtubule-associated protein 1 light chain 3(LC-3Ⅱ)were detected by quantitative reverse transcription-polymerase chain reaction and Western blot analysis.RESULTS:Compared with the normal group and the model group,the relative mRNA expression levels of autophagy-related genes(Beclin-1,ATG-5 and LC-3Ⅱ)in the experimental group were notably increased(P<0.05,P<0.01),and with the extension of treatment time,it had an increasing trend(48 h was more obvious),which showed a certain time dependency;the protein expression levels of autophagy-related genes(Beclin-1,ATG-5,and LC-3Ⅱ)were significantly increased in the experimental group(P<0.05,P<0.01).With the prolongation of treatment time,there was an increasing trend(48 h was relatively obvious),and it revealed a certain time dependency CONCLUSION:The Qingguang'an medicated serum could up-regulate autophagy related genes(Beclin1,ATG5,and LC3Ⅱ)in the TGF-β1-activated HTFs.