Protective effect of Wuzi Yanzong recipe on testicular dysfunction through inhibition of germ cell apoptosis in ageing rats via endoplasmic reticulum stress.

It has been demonstrated that excessively activated endoplasmic reticulum stress (ERS) is closely associated with ageing-related diseases and male reproductive dysfunction. Wuzi Yanzong recipe (WZ) is a classical Traditional Chinese Medicine prescription for treatment of male reproductive system diseases. However, it remains unknown whether WZ improves testicular dysfunction with ageing via ERS. In this study, we investigated the protective effects and its mechanism of WZ on testicular dysfunction in ageing rats. The results showed that treatment with WZ for 4 months significantly increased the testicular weight and index, sperm count and viability, and the levels of testosterone and decreased the levels of estradiol. In addition, WZ significantly activated the onset of ERS and prevented germ cell apoptosis by upregulating the expression levels of ERS-responsive proteins GRP78, phospho-PERK, phospho-eIF2α, ATF4, phospho-IRE-1α, XBP1 and ATF6α, and downregulating the expression levels of pro-apoptotic proteins p-JNK, Caspase12 and CHOP in testicular germ cell of ageing rats. Besides, WZ significantly decreased the numbers of TUNEL-positive cells. Taken together, WZ effectively improves ageing-related testicular dysfunction through inhibition of germ cell apoptosis via ERS.

Panax notoginseng saponins attenuate cardiomyocyte apoptosis through mitochondrial pathway in natural aging rats.

Panax notoginseng saponins (PNS) have been widely used in the cardiovascular system for the treatment of cardiovascular diseases and stroke in China. In this study, we investigated the anti-apoptotic effect of PNS on cardiomyocytes in the natural aging rat and explored the potential mechanisms regarding oxidative stress and mitochondrial function signaling pathways. Male Sprague-Dawley rats were randomly divided into five groups: adult control (3-month old), aging control (24-month old), and different doses of PNS-treated aging rat groups (10, 30, 60 mg/kg/day, orally). After treatment of PNS or saline for 6 months, the effects of PNS on the cardiomyocytes were evaluated. Results showed that PNS significantly improved the morphological changes in myocardium, prevented the increase of cardiomyocyte apoptosis in the aging rats, and improved mitochondrial dysfunction associated aging in a dose-dependent manner. PNS also significantly reversed the down-regulation of FoxO3a and Mn-SOD and up-regulated PGC-1α, LC3ß, and Beclin-1 levels. Our data demonstrated that during aging, mitochondrial dysfunction caused an increase of oxidative damage, which played a key role in cardiomyocyte apoptosis. PNS exerted an anti-apoptotic effect via attenuating oxidative damage through oxidative stress- and mitochondrial function-related signaling pathways.

Vaccine adjuvant ginsenoside Rg1 enhances immune responses against hepatitis B surface antigen in mice.

The adjuvant effect of ginsenoside Rg1 on immune responses against hepatitis B surface antigen (HBsAg) in mice was investigated. Female BALB/c mice were subcutaneously injected with saline or HBsAg antigen with or without Rg1 on days 7 and 21. Samples were collected 2 weeks after the boosting for the detection of anti-HBsAg immunoglobulin G (IgG) isotypes in sera and gamma interferon (IFN-γ) and interleukin-4 (IL-4) produced in splenocytes. The innate and adaptive immune responses were measured in mice immunized as described above. The results showed that ginsenoside Rg1 had adjuvant properties in stimulating IgG, splenocyte proliferation, and mRNA expression of cytokines IFN-γ and IL-4, as well as the expression of cell surface marker TLR4 in the HBsAg-immunized mice. These results indicate that Rg1 enhances both Th1 (IgG2b and IFN-γ) and Th2 (IgG1 and IL-4) responses. In addition, the TLR4 signaling pathway is involved in the adjuvant activities of ginsenoside Rg1.

Chikusetsu saponin V attenuates H2O2-induced oxidative stress in human neuroblastoma SH-SY5Y cells through Sirt1/PGC-1α/Mn-SOD signaling pathways.

Oxidative stress plays a vital role in the pathogenesis of neurodegenerative diseases. Chikusetsu saponin V (CsV), the most abundant member of saponins from Panax japonicus (SPJ), has attracted increasing attention for its potential to treat neurodegenerative diseases. However, the mechanisms are unclear. Our study intended to investigate the antioxidative effects of CsV in human neuroblastoma SH-SY5Y cells. Our data showed that CsV attenuated H2O2-induced cytotoxicity, inhibited ROS accumulation, increased the activities of superoxide dismutase (SOD) and GSH, and increased mitochondrial membrane potential dose-dependently. Further exploration of the mechanisms showed that CsV exhibited these effects through increasing the activation of oxidative-stress-associated factors including Sirt1, PGC-1α, and Mn-SOD. Moreover, CsV inhibited H2O2-induced down-regulation of Bcl-2 and up-regulation of Bax in a dose-dependent manner and, thus, increased the ratio of Bcl-2/Bax. In conclusion, our study demonstrated that CsV exhibited neuroprotective effects possibly through Sirt1/PGC-1α/Mn-SOD signaling pathways.

[Effects of the Saponins from Panax japonicus on Intestinal Epithelial Tight Junction Proteins in Aging Rats].

Objective: To investigate the moderating effects of total saponins of Panax japonicus on intestinal epithelial tight junction proteins in aging rats,and to explore the potential mechanism. Methods: SD rats were divided into adult group( 6 months),old model group( 24 months),and different doses( 10,30 and 60 mg / kg) of total saponins of Panax japonicus treatment groups. Levels of tight junction proteins( Occludin and ZO-1),anti-oxidative pathway proteins( Nrf2、HO-1 and NQO-1),mitochondrial biogenesis related proteins( Sirt1 and PGC-1α) and p-AMPK in the ileum were determined by immunohistochemistry staining. Results: Compared with adult group,the expressions of Occludin,ZO-1,Nrf2,HO-1,NQO-1,Sirt1 and PGC-1α of aging rats were obviously decreased( P < 0. 01),and p-AMPK was inhibited in the ileum of aging rats. Compared with aging model rats,total saponins of Panax japonicus increased the expressions of Occludin,ZO-1,Nrf2,HO-1,NQO-1,Sirt1 and PGC-1α( P < 0. 05 or P < 0. 01),and activated p-AMPK in the ileum of aging rats. Conclusion: The decreased level of intestinal tight junction proteins in the ileum of aging rats may be related to oxidative stress. Total saponins of Panax japonicus can up-regulate the level of intestinal tight junction proteins to improve the intestinal mucosal barrier dysfunction in the ileum of aging rat via reducing the levels of oxidative stress.

Sphingosine kinase-1 protects differentiated N2a cells against beta-amyloid25-35-induced neurotoxicity via the mitochondrial pathway.

Although the etiology of Alzheimer's disease (AD) is not fully understood, multiple lines of evidence suggests the importance of amyloid-ß (Aß) in the initiation/progression of the disease. Aß has been shown to induce neuronal apoptosis via the sphingomyelin/ceramide pathway. This study was designed to elucidate whether the sphingosine kinase-1 (SPK1), a critical regulator of the ceramide/sphingosine 1-phosphate rheostat, plays a pivotal role in the regulation of death and survival of differentiated neuro-2a cells in response to beta-amyloid peptide fragment 25-35 (Aß25-35). These results show that the expression of SPK1 was markedly decreased in Aß25-35-induced neurotoxicity, as evidenced by the decreased cell viability and the increased apoptotic rate. Overexpression of SPK1 significantly attenuated Aß25-35-induced neurotoxicity, whereas silencing the expression of SPK1 exacerbated it. Moreover, overexpression of SPK1 can significantly attenuate Aß25-35-induced upregulation of Bax and rehabilitate the level of Bcl-2; concomitantly, it can ameliorate mitochondrial ultrastructure. These studies demonstrate that overexpression of SPK1 may moderate Aß25-35-induced neurotoxicity by regulating the Bcl-2/Bax ratio and improving mitochondrial ultrastructure. Based on these findings, SPK1 is a potential therapeutic target for AD.

Chikusetsusaponin V inhibits inflammatory responses via NF-κB and MAPK signaling pathways in LPS-induced RAW 264.7 macrophages.

Excessive activation of macrophages is implicated in various inflammation resulted injuries. Saponins from Panax japonicus (SPJ) have been shown to possess anti-inflammatory activities. However, whether Chikusetsusaponin V (CsV), the most abundant component of SPJ, can exert anti-inflammatory activities is unknown. The present study was aimed to investigate the anti-inflammatory effects of CsV in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells and the underlying mechanisms. Our data showed that CsV dose-dependently inhibited NO, iNOS, TNF-α and IL-1ß expressions in LPS-stimulated RAW264.7 cells. Increased protein levels of nuclear NF-κB and elevated phosphorylation levels of ERK and JNK in LPS-stimulated RAW 264.7 cells were also found downregulated by CsV treatment. Furthermore, the increase of CD14 and TLR4 mRNA expression due to LPS stimulation were significantly reversed by CsV treatment. These results suggested that CsV attenuated LPS-induced inflammatory responses partly via TLR4/CD14-mediated NF-κB and MAPK pathways.

Chikusetsu saponin V attenuates MPP+-induced neurotoxicity in SH-SY5Y cells via regulation of Sirt1/Mn-SOD and GRP78/caspase-12 pathways.

Studies have shown that saponins from Panax japonicus (SPJ) possess neuroprotective effects. However, whether Chikusetsu saponin V (CsV), the most abundant member of SPJ, can exert neuroprotective effects against 1-methyl-4-phenylpyridinium ion (MPP+)-induced cytotoxicity is not known. In this study, we aimed to investigate the neuroprotective effects of CsV on MPP+-induced cytotoxicity in human neuroblastoma SH-SY5Y cells and explore its possible mechanisms. Our results show that CsV attenuates MPP+-induced cytotoxicity, inhibits ROS accumulation, and increases mitochondrial membrane potential dose-dependently. We also found that levels of Sirt1 protein and Mn-SOD mRNA significantly decreased in MPP+-treated group but were restored with CsV treatment in a dose-dependent manner. Furthermore, GRP78 protein and Caspase-12 mRNA levels were elevated by MPP+ exposure but reversed by CsV treatment. CsV inhibited the MPP+-induced downregulation of Bcl-2 and up-regulation of Bax in a dose-dependent manner and, thus, increased the ratio of Bcl-2/Bax. Overall, these results suggest that Sirt1/Mn-SOD and GRP78/Caspase-12 pathways might be involved in the CsV-mediated neuroprotective effects.

[Protective Effect of Total Flavonoids of Epimedium on D-galactose-induced Senescence in H9c2 Cells].

OBJECTIVE: To investigate the protective effect of Total Flavonids of Epimedium (TFE) on D-galactose-induced H9c2 cells senescence and to explore the underlying mechanisms. METHODS: D-galactosed (50 mmol/L) were used to induce the aging of H9c2 cells. Different concentrations of TFE (5,25 and 50 µg/mL) were added into the medium before the use of D-galactosed. Cells senescence was identified by ß-galactosidase and the changes in cell morphology were observed. The activity of superoxide dismutase(SOD) and the content of malondial dehyde (MDA) were measured. Reactive oxygen species( ROS) was observed by DCFH-DA detection and the apoptosis was tested by Hochest. RESULTS: In the D-gal group,the number of ß-galactosidase-positive cells, the content of MDA and the fluorescence intensity of ROS were markedly increased. In the meanwhile,the chromatin was condensated and the apoptotic bodies were seen by using Hochest. Compared with the D-gal group, TFE apparently decreased the quantity of ß-galactosidase-positive cells, the content of MDA, and the florescence intensity of ROS, increased the activity of SOD, and enhanced the condition of chromatin. CONCLUSION: TFE can confront D-galactose-induced senescence in H9c2 cells by increasing the ability of antioxidant and reducing the number of apoptosis.

Changes of Colon in Rats with Different Ages in Response to Lipopolysaccharide.

BACKGROUND: Lipopolysaccharide (LPS) is an endotoxin that causes inflammation, and the content of LPS increases gradually during the process of aging. Whether the response of the colon to LPS stimulation will increase with age is yet unknown. OBJECTIVE: The study investigated the effects of LPS stimulation on the colon of adult and aging rats. METHOD: 43 healthy male SD rats were divided into 4 different groups: adult group and LPS-stimulated adult group at the age of 4 months, and aging group and LPS-stimulated aging group at the age of 22 months. Rats were stimulated by intraperitoneal injection of LPS (1mg/kg) for 24 h. The morphological changes of the colon were observed, and intestinal inflammatory response, tight junction proteins, apoptosis, and proliferation in intestinal epithelial cells were detected. RESULTS: A series of morphology changes occurred in the colon of adult rats after LPS stimulation, the higher inflammatory response (TLR4, NF-κB, and IL-1ß), changes in the protein levels of tight junctions (ZO-1, Claudin1, and Claudin2), and increased apoptosis (Bax, Bcl2) and proliferation (PCNA) of intestinal epithelial cells. The above changes were also found in aging rats. LPS stimulation further promotes the above changes to some extent in the colon of aging rats. CONCLUSION: A series of colon changes in rats was significantly damaged during LPS stimulation and aging, and these changes were further aggravated to some extent in LPS-stimulated aging rats.

Relief Effects of Icariin on Inflammation-Induced Decrease of Tight Junctions in Intestinal Epithelial Cells.

Inflammatory cytokines including TNF-α and IL-1ß impair intestinal barrier function in aging by disrupting intestinal tight junction integrity. Icariin (ICA) has a variety of pharmacological effects. Indeed, ICA produces anti-inflammatory, anti-oxidative stress, and inhibitory effects on microRNA (miRNA) expression. This study was to explore whether ICA could alleviate inflammation-associated intestinal barrier function impairment in aging and its underlying mechanism. Of particular interest, network pharmacology prediction indicated the potential therapeutic impacts of ICA for the treatment of colitis. Then, rats were used to study whether ICA has a protective effect on the reduction of tight junctions caused by inflammatory cytokines. Next, Caco-2 cell monolayers were used to explore the mechanism by which ICA alleviates the down-regulation of tight junctions. Network pharmacology prediction revealed that ICA alleviated colitis via suppressing oxidative stress. After ICA intervention, expressions of inflammatory cytokines were reduced, but tight junctions, antioxidant enzymes in aging rats were up-regulated. ICA reversed the TNF-α-induced decrease in abundance of Occludin protein in Caco-2 cell monolayers. Meanwhile, ICA alleviated the increase in permeability and expression of miR-122a. However, the protective effect of ICA was markedly attenuated after transfection with miR-122a mimics. In conclusion, ICA reduced the expressions of Occludin, Claudin1, and Claudin5 in colon, which were related to the reduction of TNF-α and IL-1ß and alleviation of colonic in vivore. And ICA attenuated TNF-α-induced Occludin disruption and epithelial barrier impairment by decreasing miR-122a expression in Caco-2 cell monolayers.

Changes in the tight junctions of the testis during aging: Role of the p38 MAPK/MMP9 pathway and autophagy in Sertoli cells.

Impaired tight junction (TJ) function and autophagy and the activated p38 mitogen-activated protein kinase (MAPK)/matrix metalloproteinase 9 (MMP9) pathway in Sertoli cells cause spermatogenic disorders. However, it is unclear whether reduced TJ barrier function and autophagy and the activated p38 MAPK/MMP9 pathway in Sertoli cells are closely associated with age-related testicular dysfunction. Thus, we evaluated these changes in Sertoli cells using 6-, 12-, 18-, and 24-month-old Sprague-Dawley rats. The results showed that testicular morphology gradually degenerated, as evidenced by increased exfoliated germ cells, decreased seminiferous tubule diameter and seminiferous epithelium height, and reduced the numbers of spermatogonia, primary spermatocytes and spermatids during the process of aging. In addition, the TJs formed by adjacent Sertoli cells were progressively destroyed accompanied by an abnormal ultrastructure and decreased expression of the TJ proteins zonula occludens-1 (ZO-1), occludin, and claudin-11 with aging. Furthermore, the expression of phosphorylated p38MAPK and MMP-9 in Sertoli cells and testis gradually increased, and the expression of occludin co-localizated with MMP-9 progressively decreased. Meanwhile, autophagy levels also gradually decreased, including decreased autophagic vacuole formation and weak expression of light chain 3 (LC3) and autophagy-related 5 (Atg5) in Sertoli cells. Taken together, our results indicate that aging causes impaired TJ barrier function and degeneration of seminiferous tubules. The mechanism might be related to the activated p38MAPK/MMP9 pathway and inactivated autophagy in Sertoli cells.

Icariin attenuates perfluorooctane sulfonate-induced testicular toxicity by alleviating Sertoli cell injury and downregulating the p38MAPK/MMP9 pathway.

Perfluorooctane sulfonate (PFOS) is widely recognized as causing Sertoli cell injury and testicular toxicity in males. Icariin is a flavonoid from Epimedium, which effectively improves spermatogenesis disturbance induced by several factors in clinic. However, it is unclear whether icariin improves PFOS-induced testicular toxicity. In vivo, fifty-two male mice were randomly separated into four groups: normal control group, model group, and low and high doses of icariin-treated groups, with 13 mice in each group. Except for the normal control group, the mice in the model group and icariin-treated groups were administered PFOS (10 mg kg-1) by gavage daily for 28 consecutive days, and concurrently treated with a diet containing different doses of icariin (0, 5 or 20 mg kg-1). In vitro, TM4 cells were treated with 150 µM PFOS to induce Sertoli cell injury, and were then utilized for icariin treatment. Our results demonstrated that icariin attenuated PFOS-induced testicular toxicity by increasing the testicular, epididymal and seminal vesicle weights, epididymal and seminal vesicle indices, sperm parameters, and seminiferous epithelium height. In addition, icariin improved the PFOS-induced blood-testis barrier (BTB) disruption by alleviating the Sertoli cell junctional injury, but without affecting Sertoli cell numbers in the testis of mice. Moreover, icariin increased the expression levels of tight junction proteins (ZO-1, Occludin and Claudin-11) and gap junction proteins (CX43 and p-CX43), and decreased the expression levels of p-p38MAPK and matrix metalloproteinase 9 (MMP9) both in vivo and in vitro. Furthermore, alleviation of the Sertoli cell injury by icariin exerted similar effects as SB203580 (an inhibitor of p38MAPK) in TM4 cells. This study revealed that icariin effectively reduces PFOS-induced testicular toxicity by alleviating the Sertoli cell injury and downregulating the p38MAPK/MMP9 pathway, indicating that icariin may be an attractive dietary supplement for the intervention of PFOS-induced testicular dysfunction.

The dynamic changes of Nrf2 mediated oxidative stress, DNA damage and base excision repair in testis of rats during aging.

Accumulation of oxidative stress, DNA damage and impaired DNA repair appear to play critical roles in the decline of testicular function with aging. However, when those factors begin to lose control in testis during aging has not yet been well understood. This study was designed to assess the changes of oxidative stress and DNA damage status, and DNA repair capacity in testis during aging. Thus, male Sprague-Dawley rats at 3, 9, 15 and 24 months of age were used to delineate the dynamic changes in testicular weight and index, testosterone concentration, testicular histology, Nrf2-mediated oxidative stress, DNA damage, DNA repair and apoptosis. Results showed that testicular weight and index, testosterone concentration and spermatid number progressively declined from 9 to 24 months of age. Similarly, seminiferous tubule diameters and seminiferous epithelium heights gradually diminished with aging. Nrf2-mediated antioxidant defense ability was significantly impaired in testis with increasing age including decreased the activity of SOD and the expression levels of Nrf2, HO-1 and NQO-1, and increased the contents of MDA. In addition, DNA damage including DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) also progressively increased accompanied by increased levels of 8-hydroxydeoxyguanosine (8-OHdG) and γ-H2AX, and activated ATM/Chk2 and ATR/Chk1 pathway. Consistent with the results of Nrf2 pathway, the expression levels of APE1, OGG1 and XRCC1 involved in base excision DNA repair (BER) pathway increased from 3 to 9 months of age, and then gradually decreased after 9 months of age. Finally, TUNEL and Western blot results further confirmed germ cell apoptosis progressively increased from 3 to 24 months of age as evidenced by decreased ratio of Bcl-2/Bax and levels of Bcl-2 expression, and increased Bax expression levels. Taken together, our results suggest that downregulation of antioxidant ability mediated by Nrf2 pathway and impairment of BER capacity might correlate with increased DNA damage, and then induce declining testicular function during aging after adult.

Protective effects of saponins from Panax japonicus on neurons of the colon myenteric plexus in aging rats through reduction of α-synuclein through endoplasmic reticulum stress.

AIM: The enteric nervous system degenerates gradually with age, and α-synuclein (α-syn) is a suitable marker of enteric nervous system degeneration, which is intimately related with endoplasmic reticulum stress and unfolded protein response (UPRER ). Saponins from Panax japonicus (SPJ) have obvious protective effects on neurons in several degenerative disease models. Here, the study was designed to investigate whether SPJ could reverse the neuron degeneration through regulating the UPRER in the colon myenteric plexus of aging rats. METHODS: Aging rats had been treated with SPJ for 6 months since they were aged 18 months. Then, the colon samples were collected and neuron morphology in the myenteric plexus was observed. Immunohistochemistry staining was used to detect the expressions of NeuN, α-syn, GRP78 and three different UPRER branches. Double immunofluorescence was used to determine the co-localization of α-syn and NeuN, GRP78 and NeuN. RESULTS: Neurons degenerated in the colon myenteric plexus of aging rats, but co-localization of α-syn and NeuN increased. In addition, both the expressions of GRP78 and three UPRER branch signaling pathway proteins decreased in the colon myenteric plexus of aging rats. Treatment of SPJ almost alleviated the above effects in aging rats, except for ATF6. CONCLUSIONS: SPJ could reverse the neuron loss caused by accumulation of α-syn in the myenteric plexus of colon in aging rats, which is potentially associated with increased GRP78 and most URPER changes. Geriatr Gerontol Int 2021; 21: 85-93.

Panax notoginseng Saponins Attenuate Neuroinflammation through TXNIP-Mediated NLRP3 Inflammasome Activation in Aging Rats.

INTRODUCTION: Microglia-mediated inflammatory responses play a crucial role in aging-related neurodegenerative diseases. The TXNIP/NLRP3 pathway is a key pathway leading to microglial activation. Panax notoginseng Saponins (PNS) have been widely used for the treatment of stroke in China. OBJECTIVE: This study evaluates the anti-neuroinflammatory effect of PNS and investigates the mechanism via TXNIP-mediated NLRP3 inflammasome activation in aging rats. MATERIAL AND METHODS: Eighteen-month-old Sprague-Dawley rats were randomly divided into the aging control group and PNS treated groups (n=15 each group). For PNS-treated groups, rats were administrated food with PNS at the doses of 10 mg/kg and 30 mg/kg for consecutive 6 months until they were 24-month old. Rats from the aging control group were given the same food without PNS. Twomonth- old rats were purchased and given the same food until they were 6-months old as the adult control group (n = 15). Then, the cortex and hippocampus were rapidly harvested and deposited. H&E staining was used to assess histo-morphological changes. Western blotting was carried out to detect the protein expression. Immunofluorescence was employed to measure the co-localization of NLRP3, TXNIP and Iba-1. In vitro model was established by LPS+ATP co-incubation in the BV2 microglia cell line. RESULTS: Aging rats exhibited increased activation of microglia, accompanied by a high level of IL-1ß expression. Meanwhile, aging rats showed enhanced protein expression of TXNIP and NLRP3 related molecules, which co-localized with microglia. PNS treatment effectively reduced the number of degenerated neurons and reversed the activation of the TXNIP/NLRP3 inflammatory pathway. In vitro results showed that PNS up to 100 µg/ml had no significant toxicity on BV2 microglia. PNS (25, 50 µg/ml) effectively reduced the inflammatory response induced by LPS and ATP co-stimulation, thus inhibiting the expression of TXNIP/NLRP3 pathway-related proteins. DISCUSSION AND CONCLUSION: PNS treatment improved aging-related neuronal damage through inhibiting TXNIP mediated NLRP3 inflammasome activation, which provided a potential target for the treatment of inflammation-related neurodegenerative diseases.

Changes of Wnt/ß-catenin signalling, BMP2, and BMP4 in the jejunum during ageing in rats.

BACKGROUND AND STUDY AIMS: The renewal of intestinal epithelium is maintained by intestinal stem cells (ISCs). Studies have found an age-dependent increase of Esg+/Dl+ progenitor cells in the midgut of Drosophila. However, changes of ISCs and the molecular regulation in mammalian animals with age are yet unknown. The aim of this study was to find out the changes of ISCs and molecular regulation in mammalian animals during the process of ageing. MATERIAL AND METHODS: Thirty Sprague-Dawley rats were divided into three groups: young (3 months old), adult (6 months old), and ageing (24 months old). Levels of PCNA, Bmi1, ß-catenin and BMP4 were examined by Immunohistochemistry staining. Levels of Bmi1, GSK-3ß, Dkk1 and BMP2 were determined by Western Blot. RESULTS: Our results showed that the proliferation of ISCs was decreased and the number of intestinal stem cells declined in ageing rats. The niches of ISCs, including Wnt signalling pathway and some proteins of Bone morphogenetic protein (BMP) signalling pathway, were downregulated in the jejunum of ageing rats. CONCLUSION: Our study indicated that age-related decreased proliferation of intestinal stem cells in the jejunum could be associated with the alleviation of niches, including Wnt signalling pathway and some proteins of BMP signalling pathway.

Saponins of Panax japonicus Confer Neuroprotection against Brain Aging through Mitochondrial Related Oxidative Stress and Autophagy in Rats.

BACKGROUND: Oxidative stress and mitochondrial dysfunction play a vital role in the pathogenesis of brain aging. Saponins from Panax japonicus (SPJ) have attracted much attention for their potential to attenuate age-related oxidative stress as the main ingredient in rhizomes of Panax japonicus. OBJECTIVE: This study aimed to investigate the neuroprotective effects of SPJ on natural aging rats as well as the underlying mechanisms regarding oxidative stress and mitochondrial pathway. METHODS: Sprague-Dawley rats were divided into control groups (3-, 9-, 15- and 24-month old groups) and SPJ-treated groups. For SPJ-treated groups, SPJ were orally administrated to 18-month old rats at doses of 10 mg/kg, 30 mg/kg and 60 mg/kg once daily. Control groups were given the same volume of saline. After the treatment with SPJ or saline for six months, the cortex and hippocampus were rapidly harvested and deposited at -80°C after the rats were decapitated under anesthesia. The neuroprotective effects of SPJ were estimated by histopathological observation, TUNEL detection, biochemical determination and western blotting. RESULTS: SPJ improved pathomorphological changes in neuronal cells and decreased apoptosis in the cortex and hippocampus of aging rats, increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), Na+/K+-ATPase, Ca2+-ATPase and Ca2+/Mg2+-ATPase whereas, decreased malondialdehyde (MDA) contents in the cortex of aging rats. Furthermore, the SPJ increased silent mating type information regulation 2 homolog-1 (SIRT1) protein expression, decreased acetylated level of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in the cortex and hippocampus of aging rats, and reversed the aging-induced decline of Forkhead box O3 (Foxo3a), Superoxide Dismutase 2 (SOD2), microtubule-associated protein light chain 3 (LC3II) and Beclin1 levels in the cortex and hippocampus. CONCLUSION: Our data showed that SPJ conferred neuroprotection partly through the regulation of oxidative stress and mitochondria-related pathways in aging rats.

Icariin Improves Age-Related Testicular Dysfunction by Alleviating Sertoli Cell Injury via Upregulation of the ERα/Nrf2-Signaling Pathway.

Sertoli cells play crucial roles in spermatogenesis and are impaired by aging. Icariin, a flavonoid from Epimedium, has been reported to exhibit anti-aging effects and improve testicular dysfunction in the clinical setting. However, whether icariin improves age-related degeneration of testicular function via protection from Sertoli cell injury remains unclear. In the present study, we evaluated the protective effect of icariin on Sertoli cell injury and explored the possible mechanism(s) in vivo and in vitro. Dietary administration of icariin for 4 months significantly ameliorated the age-related decline in testicular function by increasing testicular and epididymal weights and indices, sperm count and sperm viability, testicular testosterone and estradiol concentrations, and seminiferous tubule diameters and heights. In addition, icariin protected age-related Sertoli cells from injury as evidenced by an analysis of Sertoli cell number, ultrastructure, and function. Such changes were accompanied by upregulation of ERα and Nrf2 signaling in Sertoli cells. Parallel in vitro studies also demonstrated that icariin inhibited untoward effects on the TM4 mouse Sertoli cell line with concomitant upregulation of ERα and Nrf2 signaling. Conversely, ERα siRNA reversed icariin-mediated protection of Sertoli cell injury. Our data suggest that icariin effectively ameliorates age-related degeneration of testicular function by alleviating Sertoli cell injury via the ERα/Nrf2 signal-transduction pathway. Thus, mitigating Sertoli cell damage via the ERα/Nrf2 signaling pathway likely represents a promising strategy for the prevention of age-related testicular dysfunction.

Distinct expression patterns of aging effects on the UPRER signaling pathway in rat colon and regulatory role of saponins from Panax japonicus.

BACKGROUND: Studies have reported that the unfolded protein response of ER (UPRER) declines in the several organs of aging mice. However, changes of UPRER during the aging process in the intestine are rarely reported. Our previous studies have demonstrated that Saponins from Panax japonicus (SPJ) have anti-aging effects in different murine models and can modulate ER stress. In the present study, we focused on age-dependent expressions of UPRER in the intestine of 6- to 24-month-old rats by an immunohistochemical (IHC) method and determined whether SPJ could regulate the three different UPRER branches of the colon in aging rats. METHODS: Aging rats had been treated with different doses (10 and 30 mg/kg) of SPJ for 6 months, which were mixed with feed, since they were 18 months old. Then the expressions of GRP78 and three different UPRER branches were determined by immunohistochemistry (IHC). Results: Total expressions of GRP78 and p-JNK increased, and other UPRER proteins decreased in the colon of aging rats, while SPJ treatment relieved the corresponding changes in aging rats. Here we also found different patterns of GRP78 and the three UPRER branches in the different layers of colon in rats. CONCLUSION: The study demonstrated that UPRER declined and GRP78 increased in the colon of aging rats. SPJ could reverse most URPER changes in the colon of aging rats. This study also showed different expression patterns of the three branches of UPRER in different layers of the colon in rats.