Guiqi Dingnian Prescription Delays Senescence of Mouse Mesangial Cells by Regulating JAK2/STAT Pathway Activity / 中国实验方剂学杂志

Objective:To investigate the effects of Guiqi Dingnian prescription （GDP） on the expression of related molecules in Janus tyrosine kinase 2 （JAK2）/signal transducer and activator of transcription （JAK2/STAT） signaling pathway of <italic>D</italic>-galactose （<italic>D</italic>-gal）-induced senescent mesangial cells. Method:The senescent mouse mesangial cells induced by 10 g·L^-1 <italic>D</italic>-gal were continuously treated with 40 mg·L^-1GDP for three days. The senescence of the treated cells was determined by senescence-associated （SA）-<italic>β</italic>-gal staining. The cell cycle was detected by flow cytometry. The cell viability was analyzed using the cell counting kit-8 （CCK-8）. The mRNA expression levels of tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-6 （IL-6）， nuclear transcription factor-<italic>κ</italic>B （NF-<italic>κ</italic>B）， and IL-1<italic>α</italic> were detected by real-time polymerase chain reaction （Real-time PCR）. The protein expression levels of STAT1， phosphorylated STAT1 （p-STAT1）， STAT3， and p-STAT3 in the JAK2/STAT signaling pathway were determined by Western blot. Result:CCK-8 results showed that the optimal concentration of GDP was 40 mg·L^-1. Compared with the blank group， the positive rate of SA-<italic>β</italic>-gal in the model group was significantly higher（<italic>P</italic><0.01）， the percentage of cells in G₀/G₁ phase was significantly increased（<italic>P</italic><0.05）， the percentage of cells in G₂/M and S phase was significantly decreased（<italic>P</italic><0.01）. The mRNA expression levels of TNF-<italic>α</italic>，IL-6，NF-<italic>κ</italic>B and IL-1<italic>α </italic>were significantly increased（<italic>P</italic><0.01）. Compared with the model group， the model + GDP group exhibited significantly decreased SA-<italic>β</italic>-gal-positive cells （<italic>P</italic><0.05）， reduced cells in the G₀/G₁ phase （<italic>P</italic><0.05）， increased cells in the G₂/M and S phases （<italic>P</italic><0.01）， and down-regulated TNF-<italic>α</italic>， IL-6， NF-<italic>κ</italic>B， and IL-1<italic>α </italic>mRNA expression （<italic>P</italic><0.05） and STAT1， p-STAT1， STAT3， and p-STAT3 protein expression （<italic>P</italic><0.05）. Conclusion:GDP delays the senescence of mouse mesangial cells possibly by down-regulating the expression of related molecules in the JAK2/STAT pathway.

Effect of ginsenoside Rg_1 in delaying premature ovarian failure induced by D-gal in mice through PI3K/Akt/mTOR autophagy pathway / 中国中药杂志

The aim of this paper was to study the role of phosphoinositide 3-kinase(PI3 K), protein kinase B(Akt) and mamma-lian target of rapamycin(mTOR) in the inhibition of premature ovarian failure induced by D-galactose(D-gal) in mice model by ginsenoside Rg_1(Rg_1). Fifty-four female SPF BALB/c mice were randomly divided into PBS group, D-gal group, and Rg_1 group. In the D-gal group, D-galactose(200 mg·kg~(-1)·d~(-1)) was injected subcutaneously into the neck and back for 42 days. In the PBS group, an equal amount of phosphate buffered saline(PBS) was injected into the neck and back for 42 days. In addition to the therapy of D-gal group, Rg_1 group was given Rg_1(20 mg·kg~(-1)·d~(-1)) through intraperitoneal injection since the 15 th day for 28 days, at the same time, the D-gal group and the PBS group were also given an equal amount of PBS through intraperitoneal injection since the 15 th day for 28 days. After the treatment, the estrous cycle changes of the mice were detected, and the ovarian SA-β-Gal staining was used to detect the changes of ovarian aging. Western blot was used to detect the changes in protein expressions of PI3 K, Akt, mTOR, S6 k, LC3-Ⅱ and P16~(INK4 a). Fluorescence quantitative PCR was used to detect the changes in mRNA expressions of PI3 K, Akt, mTOR, S6 k, LC3-Ⅱ and P16~(INK4 a). According to the findings, compared with the PBS group, the D-gal group began to show estrous cycle disorder in the 3 rd week,the ovarian SA-β-Gal staining positive granulosa cells increased in the D-gal group, the expression of senescence marker P16~(INK4 a) increased, while the expression of autophagy signaling molecule LC3-Ⅱ decreased. After treatment with Rg_1, the positive rate of ovarian SA-β-Gal staining in Rg_1 group decreased, the expression level of autophagy signaling molecule LC3-Ⅱ in Rg_1 group was higher than that in D-gal group, while the expression level of senescence marker P16~(INK4 a) was lower than that in D-gal group. Compared with the PBS group, the protein and mRNA expressions of PI3 K, Akt, mTOR and S6 k in the D-gal group were up-regulated, the protein expressions of Akt, mTOR and S6 k in the Rg_1 group were up-regulated, and the mRNA expressions of PI3 K and mTOR were up-regulated. After treatment with Rg_1, the protein expressions of PI3 K, Akt, mTOR and S6 k in the Rg_1 group were lower than those in the D-gal group, while the mRNA expressions of Akt, mTOR and S6 k in the Rg_1 group were lower than those in the D-gal group. The finding ssuggested that Rg_1 has the effect in delaying ovarian premature failure in D-gal-induced mouse models, and PI3 K/Akt/mTOR autophagy signaling pathways play an important role.

Senescence induced by D-galactose and its biological mechanism in rat bone marrow stromal cells / 基础医学与临床

Objective To establish an aging model of rat bone marrow stromal cells (BMSCs) in vitro and in vivo, in order to study the senescence biology of aging BMSCs .Methods The control cell group ( in vitro):isolating, puri-fying and culturing BMSCs from healthy male SD rats .collecting the third generation ( P3) of BMSCs for analysis . The aging model group (in vitro):the P3 BMSCs were incubated with D-Galactose (D-Gal, 30 g/L) for 48 hours. The aging rat model group ( in vivo): the rats were given 120 mg D-Gal by the way of daily neck subcutaneous injection for 42 consecutive days .The control rat group ( in vivo):the rats were administrated with the same volume of saline for the same times .On the second day after the aging model was established , the BMSCs were collecting and culturing for study.1)The proliferative potency was detected by cell counting Kit-8(CCK-8);the distribution of cell cycle and apoptosis was detected by flow cytometry (FCM);2)the ratio of aging BMSCs was examined by the senescence-associated β-Galactosidase(SA-β-Gal) staining;3)malonaldehyde(MDA) content and total super-oxide dismutase(SOD) was examined activity by enzymatic assay; the level of reactive oxygen species (ROS) by DCFH-DA fluorescent staining was counted with FCM;4 ) the expression level of senescence-related signaling was proteins of P16 , P21 , P53 , CDK2 and cyclin D by Western blot .Results Compared with the matched control group, the BMSCs of aging model group displayed a significant decrease in proliferation; the BMSCs were held in G1 phase arrest as the proportion of the cells in G 1 phase increased , while that decreased in S phase ( P<0.05 );and the positive ratio of SA-β-Gal stained BMSCs also significantly increased ( P <0.05 ); BMSCs in the aging model group showed an increasing level of ROS and MDA , meanwhile a decline in total SOD activity was decreased (P<0.05);P16,P21 and P53 protein expression in aging BMSCs was obviously enhanced (P<0.05), at the same time the expression of CDK2 and cyclin D was also decreased ( P<0.05 ) .Conclusions D-Gal can be used to develope an aging model of BMSCs .It acts through up-regulation of expressions of aging-related proteins and in-hibition of oxidative stress injury and chronic inflammation .

A murine model of LPS/D-gal-induced acute hepatitis and alterations in mTOR signaling / 中国实验动物学报

Objective To explore the changes of mTOR signaling in LPS/D-gal-induced acute hepatitis in mice . Methods Twenty-six healthy adult female ICR mice were divided into two groups:the control group and experimental group, 13 mice in each group .LPS/D-gal was used to induce acute hepatitis in the mice .The survival of mice was moni-tored within 24 hours after LPS/D-gal challenge .At 6 hours after challenge , samples of serum and liver tissue were collect-ed for further analysis.Results Injection of LPS/D-gal resulted in acute death of the mice within 24 hours.At 6 hours post LPS/D-gal injection , the blood levels of ALT and AST were significantly increased .The mRNA expression of inflammatory cytokines Tnfa and Il6 was up-regulated in LPS/D-gal-induced hapatitis , in which DNA fragmentation and activation of caspase-3 were subsequently observed .Immunoblot analysis showed that both mTOR pathway and NF-κB pathway were ac-tivated.Unexpectedly , inhibition of mTOR signaling could neither decrease the apoptosis in the liver nor increase the sur -vival of mice .Conclusions The results of the present study indicate that mTOR signaling may play pleiotropic roles in the pathogenesis of LPS/D-gal-induced hepatitis .

Effects of TotalPanax Japonicus Extract onD-galactose Induced Aging Mice / 中国中医药信息杂志

Objective To research the protective effects of totalPanax Japonicus extract on learning memory, antioxidation, and anti-apoptosis of aging mice,and explore the mechanism. Methods Male mice were randomly divided into normal group, model group, Vitamin E (VE) group, Panax Japonicus extract low and high dose group. Except for the normal group, the other groups were injected withD-gal on the back of the neck subcutaneously to establish aging model. Normal group and model group were given a gavage with saline and each treatment group was given a gavage with totalPanax Japonicus extract and VE once a day for 7 weeks after the aging model established. All mice were be measured their learning and memory ability in the eighth week. After the test, the morphological changes of CA1 neurons were observed by HE stain. SOD, GSH-Px, MDA levels in brain tissue were measured by biochemical method, the expressions of Bcl-2 and Bax mRNA were evaluated by RT-PCR.Results Mice inPanax Japonicus extract low and high dose group could spend less time in searching for the platform, improve the learning and memory ability. TotalPanax Japonicus extract increased the activity of SOD and GSH-Px, while decreased the content of MDA. In addition, it could increase the expression level of Bcl-2 mRNA and reduce the expression level of Bax mRNA as well.Conclusion TotalPanax Japonicus extract has anti-aging effect.

Effects of EGB on hippocampus CA1 region Caspase-9P35 activity in dementia rats induced by Dgal / 中华行为医学与脑科学杂志

Objective To investigate the effect and mechanism of extract ginkgo biloba (EGb) on learning memory ability and hippocampus CA1 region Caspase-9P35 activity in dementia rats induced by D-gal.Methods 48 rats were randomly divided into six groups.The dementia model were established by injecting intraperitoneally with the D-gal and each EGb group was injected different doses of EGb simultaneously.TUNEL method was used to detect apoptosis of hippocampal neurons in the CA1 region.Immunohistochemistry and Western Blot were used to determine the expression of Caspase-9P35 in hippocampus CA1 region.Results Compared with the normal group,TUN EL-positive neurons ( (37.8 ± 1.3 ),(0.8 ± 0.2 ) ) and the activity of Caspase-9P35 ( (37.6 ±1.8 ),(6.2 ± 1.3 ) ) had significant increase in hippocampal CA1 subfield of D-gal group (P ＜ 0.01 ).Contrast to D-Gal group,TUNEL-positive neurons ( ( 17.6 ± 0.9),(9.8 ± 0.8 ),( 37.8 ± 1.3 ) ) and the activity of Caspase9P35( (28.6 ± 1.3),(25.0 ± 1.6),(37.6 ± 1.8) ) were significant decreased in EGb-M and EGb-H group (P＜0.01 ).While TUNEL-positive neurons and the activity of Caspase-9P35 had not significant difference in the therapy group than D-Gal group (P ＞ 0.05).Conclusion EGb can improve the cognitive level of the dementia rats.One of the therapeutic mechanisms may be to decrease the hippocampus CA1 region Caspase-9P35 activity.The results of the pretreatment group was more effective than the therapy group.

Effect of Pyrroloquinoline Quinone on Aging of Rat Hippocampal Neurons in Vitro / 中国康复理论与实践

@#Objective To investigate the effect of pyrroloquinoline quinone(PQQ) on the aging of rat hippocampal neurons induced by D-galactose(D-gal).Methods Hippocampal neurons were cultured in vitro.The aging of the hippocampal neurons was induced by high dose D-gal,PQQ protection were used 30 min before D-gal.The metamorphosis of hippocampal neurons was observed under the microscope.The contents of free radical was measured.The incidence of apoptosis of hippocampus cells was tested with the flow cytometry.The expression of Bax was detected with immunohistochemical staining.Results After the cells cultured in vitro exposed to D-gal,the content of free radical and the expression of Bax of the hippocampal neurons increased.After pretreatment of the cultured neurons with PQQ,the contents of free radical and the expression of Bax decreased,the survival of hippocampal neurons increased.Conclusion PQQ may slow the aging progress of hippocamal neurons induced by D-gal.

Effect of PQQ on the hippocampal neurons of aging rat induced by D-galactose / 基础医学与临床

Objective To investigate the effects of pyrroloquinoline quinone(PQQ) on the hippocampal neurons of aged rats induced by D-galactose(D-gal).Methods D-gal was used to induce the model of aging rat,PQQ was administered into rat lateral intracerebroventricle.After 50 days the metamorphosis of hippocampal neurons was observed by H-E and Nissl's staining.The apoptosis rate of hippocampus was tested by flow cytometry.The contents of free radical and C-FOS protern were measured.Results Compared with the control group,the size of the neurosoma was slightly changed,the optical density of Nissl's was decreased,the content of free radical and the apoptosis rate increased markedly in D-gal group.After PQQ injection with D-gal,the size of neurosoma and the optical density of Nissl's were markedly increased,the content of free radical and the apoptosis rate of hippocampus did not change.PQQ improved the expression of C-FOS protern.Conclusion PQQ can slow down the aging progress of hippocamal neurons induced by D-gal.

Effects of human fetal hepatic stimulator substance on D-galac-tosamine-induced liver necrosis in rats / 第三军医大学学报

The effects of human fetal hepatic stimulator substance (h-HSS) and liver cytosol (h-FLC) on the survival rate,the intensity of liver damage,and liver regeneration in rats with liver necrosis induced with D-galactosamine were observed.The physico-chemical properties of h-HSS were preliminarily determined.It was found that both h-HSS and H-FLC could markedly increase the.survival rate of the rats with toxic liver necrosis,significantly decrease the serum level of ornithine carbamoyl transferase and mitochondria aspartic trans-aminase,improve the hepaplastin activity,and elevate the incorporation rate of 3H-thymidine with liver DNA.h-HSS was stable after heated to 95℃ for 30 minutes,and sensitive to trypsin treatment.It demonstrated one major band at 15 000 daltons and 3 minor ones at 24 000,34 000,and 40 000 daltons respectively on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The relative content of the major band was 47.57%.

THERAPEUTIC EFFECTS OF BHGF ON ACUTE HEPATIC FAILURE INDUCED BY D-GALACTOSAMINE IN RATS / 中国药理学通报

The therapeutic effects of bovine hepatocytic growth factor ( BHGF ) were studied on acute hepatic failure ( AHF ) induced by D-galactosamine ( D-Gal ) in rats. The BHGF was isolated from the hepatic cytosol of newborn cow in our laboratory and its molecular is under 20 000 dalton. The results showed that. l)the survival rate of the rats with AHF was significantly enhanced after therapy with BHGF, 2 ) the 8H-TdR were highly incoporated into hepatocytic DNA of the rats, 3 ) the pathological damage of the rat liver was obviously improved, when comparied with the control. It is Suggested that BHGF could stimulate the hepatocytic DNA synthesis and be effective in the therapy of AHF in rats.

THE ESTABLISHMENT AND PRELIMINARY USE OF D-GALACTOSAMINE CYTOTO- XICITY MODEL OF PRIMARILY CULTURED HEPATOCYTES / 解放军医学杂志

A cytotoxicity model of primary cultured hepatocytes with D-galactosamine (D-Gal) has been established. The ALT value of culture medium was used as the marker of cell injury. Using this model, the in vitro antihepatotoxic effects of hepatic stimulator substance (HSS), isolated from regenerating adult rat liver, as well as Potenlin and 15-amino acid solution were observed. The results showed that although HSS could stimulate DNA synthesis in serum-free culture of adult rat hepatocytes, it had no effect to reduce ALT value in vitro. However the latter could be reduced by Potenlin or 15-amino acid solution in high concentrations. The described model may be useful for preliminary screening of antihepatotoxic activity of drugs.