Saponins from Panax japonicus improve neuronal mitochondrial injury of aging rats.

CONTEXT: Panax japonicus is the dried rhizome of Panax japonicus C.A. Mey. (Araliaceae). Saponins from Panax japonicus (SPJ) exhibit anti-oxidative and anti-aging effects. OBJECTIVE: We evaluated the neuroprotective effects of SPJ on aging rats. MATERIALS AND METHODS: Sprague-Dawley rats (18-months-old) were randomly divided into aging and SPJ groups (n = 8). Five-month-old rats were taken as the adult control (n = 8). The rats were fed a normal chow diet or the SPJ-containing diet (10 or 30 mg/kg) for 4 months. An in vitro model was established by d-galactose (d-Gal) in the SH-SY5Y cell line and pretreated with SPJ (25 and 50 µg/mL). The neuroprotection of SPJ was evaluated via Nissl staining, flow cytometry, transmission electron microscopy and Western blotting in vivo and in vitro. RESULTS: SPJ improved the neuronal degeneration and mitochondrial morphology that are associated with aging. Meanwhile, SPJ up-regulated the protein levels of mitofusin 2 (Mfn2) and optic atrophy 1 (Opa1) and down-regulated the protein level of dynamin-like protein 1 (Drp1) in the hippocampus of aging rats (p < 0.05 or p < 0.01 vs. 22 M). The in vitro studies also demonstrated that SPJ attenuated d-Gal-induced cell senescence concomitant with the improvement in mitochondrial function; SPJ, also up-regulated the Mfn2 and Opa1 protein levels, whereas the Drp1 protein level (p < 0.05 or p < 0.01 vs. d-Gal group) was down-regulated. DISCUSSION AND CONCLUSIONS: Further research on the elderly population will contribute to the development and utilization of SPJ for the treatment of neurodegenerative disorders.

Saponins from Panax japonicus attenuate cognitive impairment in ageing rats through regulating microglial polarisation and autophagy.

CONTEXT: Panax japonicus is the dried rhizome of Panax japonicus C.A. Mey. (Araliaceae). Saponins from Panax japonicus (SPJ) exhibit anti-inflammatory and antioxidative effects. OBJECTIVE: To explore the neuroprotective effect of SPJ on natural ageing of rat. MATERIALS AND METHODS: Sprague-Dawley (SD) rats 18-month-old were divided into ageing control, ageing treated with SPJ 10 or 30 mg/kg (n = 8). Five-month-old rats were taken as the adult control (n = 8). Rats were fed regular feed or feed containing SPJ for 4 months. Cognitive level was evaluated by Morris water maze (MWM) test. The mechanisms of SPJ's neuroprotection were evaluated by transmission electron microscope, western blot analysis, and immunofluorescence in vivo and in vitro. RESULTS: SPJ attenuated ageing-induced cognitive impairment as indicated by elevated number of times crossing the target platform (from 1.63 to 3.5) and longer time spent in the target platform quadrant (from 1.33 to 1.98). Meanwhile, SPJ improved the morphology of microglia and synapse, and activated M2 microglia polarisation including increased hippocampus levels of CD206 (from 0.98 to 1.47) and YM-1 (from 0.67 to 1.1), and enhanced autophagy-related proteins LC3B (from 0.48 to 0.82), Beclin1 (from 0.32 to 0.51), Atg5 (from 0.22 to 0.89) whereas decreased p62 level (from 0.71 to 0.45) of ageing rats. In vitro study also showed that SPJ regulated the microglial polarisation and autophagy. DISCUSSION AND CONCLUSIONS: SPJ improved cognitive deficits of ageing rats through attenuating microglial inflammation and enhancing microglial autophagy, which could be used to treat neurodegenerative disorders.

[Effect of total saponins from Panax japonicus on non-alcoholic steatohepatitis by regulating autophagy].

Non-alcoholic steatohepatitis(NASH) was induced by high-sugar and high-fat diet in mice to investigate the intervention effect of total saponins from Panax japonicus(TSPJ) and explore its possible mechanism. Mice were fed with high-sugar and high-fat diet to establish NASH model, and intervened with different doses of TSPJ(15, 45 mg·kg~(-1)). The animals were fed for 26 weeks. The histomorphology and pathological changes of liver tissues were observed by HE staining. The transcriptional expression levels of miR-199 a-5 p, autophagy related gene 5(ATG5) and inflammatory cytokines interleukin-6(IL-6), interleukin-1ß(IL-1ß) and tumor necrosis factor α(TNF-α) in mouse liver were measured by quantitative Real-time polymerase chain reaction(qRT-PCR). Western blot was used to detect the expression of autophagy-related proteins ATG5, P62/SQSTM1(P62), and microtubule-associated protein light chain 3(LC3)-I/Ⅱ proteins in mouse liver. The expression of P62 protein was detected by immunofluorescence staining. In order to verify the targeting regulation relationship between miR-199 a-5 p and ATG5, miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor were transfected into Hepa 1-6 cells, and the expression of ATG5 mRNA and protein was detected. pMIR-reportor ATG5-3'UTR luciferase reporter gene plasmid was constructed and co-transfected with miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor into Hepa 1-6 cells to detect luciferase activity. In vivo, HE staining in the model group showed typical fatty degeneration and inflammatory infiltration, with increased expression of miR-199 a-5 p and decreased expression of ATG5 mRNA and protein. The expression of autophagy-associated protein P62 increased significantly, the ratio of LC3Ⅱ/Ⅰ decreased, and the transcriptional expression of inflammatory factors increased significantly. After the intervention by TSPJ, the pathological performance of liver tissue was significantly improved, the expression of miR-199 a-5 p decreased and the expression of ATG5 mRNA and protein increased, the expression of autophagy-associated protein P62 decreased significantly, the ratio of LC3Ⅱ/Ⅰ increased, and the transcriptional expression of inflammatory cytokines IL-6, IL-1ß and TNF-α decreased significantly. In vitro, it was found that the expression of ATG5 mRNA and protein and luciferase activity decreased significantly in miR-199 a-5 p overexpression cells, while after inhibition of miR-199 a-5 p expression, the expression level of ATG5 mRNA and protein and luciferase activity increased. The results showed that TSPJ can improve NASH in mice fed with high-sugar and high-fat diet, and its mechanism may be related to the regulation of miR-199 a-5 p/ATG5 signal pathway, the regulation of autophagy activity and the improvement of inflammatory response of NASH.

[Triterpene saponins in Panax japonicus and their ~(13)C-NMR spectroscopic characteristics].

Panax japonicus( PJ) is a valuable medicinal plant belonging to the genus Panax of Araliaceae,the recumbent rhizome of which is widely used in clinic therapy,healthcare products and as cosmetic additives with functions of dissipating stasis,reducing swelling,stanching bleeding,and reinforcing deficiency,etc. PJ contains abundant levels of oleanane-and dammarane-type triterpene saponins,which are considered as the material basis for exerting pharmacodynamic action. Based on the previous researches,more than110 triterpene saponins have been reported from PJ. These triterpene saponins were summarized in this review,and could be classified into dammarenediol Ⅱ,protopanaxadiol,protopanaxatiol,ocotillol,oleanolic acid,ursolic acid and miscellaneous subtypes,according to their molecular skeletons in biosynthesis processes. Further more,the structural features of these triterpene saponins in the seven different subtypes,together with their~(13)C-NMR spectroscopic characteristics were described,hoping to provide available information for chemical diversity research of PJ.

[Improved effects of saponins from Panax japonicus on decline of cognitive function in natural aging rats via NLRP3 inflammasome pathway].

The aim of this paper was to investigate the effect of total saponins from Panax japonicus( SPJ) on cognitive decline of natural aging rats and its mechanism. Thirty male SD rats of eighteen month old were randomly divided into three groups: aged group,10 mg·kg~(-1) SPJ-treated group and 30 mg·kg~(-1) SPJ-treated group. The SPJ-treated groups were given SPJ at the dosages of 10 mg·kg~(-1) and 30 mg·kg~(-1),respectively,from the age of 18 to 24 months. Aged group were lavaged the same amount of saline,10 six-month-old rats were used as control group,with 10 rats in each group. The open field test,novel object recognition and Morris water maze were performed to detect the changes of cognitive function in each group. The changes of synaptic transmission of long-term potentiation( LTP) in hippocampal CA1 region were detected by field potential recording. Western blot was used to detect the protein levels of NLRP3,ASC,caspase-1 and the changes of Glu A1,Glu A2,CAMKⅡ,CREB and phosphorylation of CAMKⅡ,CREB in each group.The results showed that SPJ could improve the decline of cognitive function in aging rats,reduce the damage of LTP in the hippocampal CA1 region of aged rats,and decrease the expression of NLRP3,ASC,caspase-1 in aging rats. At the same time,SPJ could enhance the membrane expression of AMPA receptor( Glu A1 and Glu A2),and increase the expression of p-CAMKⅡand p-CREB in aging rats.SPJ could improve cognitive decline of natural aging rats,and its mechanism may be related to regulating NLRP3 inflammasome,thus regulating the membrane expression of AMPA receptor,and enhancing the expression phosphorylation of CAMKⅡ and CREB.

[Effect of saponins extracted from Panax japonicas on inhibiting myocardial fibrosis by TGF-ß1/Smad3 signaling pathway in aging rats].

To investigate the amelioration effect of saponins extracted from Panax japonicas (SPJ) on myocardial fibrosis in natural aging rats and its mechanisms, male SD rats aged 18 months were randomly divided into 3 groups (aging model group, low-dose SPJ group and high-dose SPJ group), with 10 rats in each group. SPJ groups were given SPJ at different doses (10, 60 mg·kg⁻¹·d⁻¹) consecutively for 6 months, meanwhile, aging model group was treated with the equal volume of saline for 6 months until 24 months old. Another 10 rats aged 6 month were used as young control group. The changes of myocardial morphological were observed by haematoxylin-eosin (HE) staining. Masson staining was used to observe the changes of collagen deposition in rat hearts. RT-PCR was used to detect the mRNA expression levels of myofibroblast marker α-SMA, collagen-related protein COL1α2, COL3α1 and matrix metalloproteinase MMP2, MMP9. Western blot was used to test the changes of the protein expressions of TGF-ß1, p-Smad3, IL-1ß and TNF-α in heart tissues. SPJ can effectively improve the arrangement of myocardial fibers, decrease inflammatory infiltration and reduce collagen deposition in aging rats. SPJ can effectively down-regulate the mRNA expression levels of COL1α2, COL3α1, α-SMA, MMP9, MMP2 and inhibit the protein expressions of TGF-ß1, p-Smad3, TNF-α, IL-1ß in the natural aging heart tissues. SPJ can effectively alleviate myocardial fibrosis in natural aging rats, and its mechanisms was related to the inhibition of the protein expressions of TGF-ß1, p-Smad3 and the reduction of myocardial inflammation in rat hearts.

[Protective effect of Wuzi Yanzong recipe on testicular germ cell apoptosis in natural ageing rats through endoplasmic reticulum stress].

To study the protective effects of Wuzi Yanzong recipe on testis germ cell apoptosis in natural ageing rats through endoplasmic reticulum stress (ERS), 16-month-old male SPF grade SD rats were randomly divided into three groups: ageing model group, and low and high-dose Wuzi Yanzong recipe groups (WZ, 1 and 4 g·kg⁻¹), with 10 rats in each group. In addition, 2-month-old SD male rats were used as adult control group. The ageing model group and the adult control group were fed with normal diet for 4 months. WZ groups were given the medicated feed for 4 months. After fasting for 12 hours, the rats were put to death. Then, the testes were immediately collected. The change of testicular tissue morphology was observed by HE staining. The expression levels of ER stress-related proteins GRP78, p-PERK, p-eif2α, ATF4, p-IRE1, XBP1, ATF6 and apoptosis-related proteins CHOP, caspase12 and p-JNK in testes were detected by Western blot. Compared with the ageing model group, Wuzi Yanzong recipe alleviated the morphological changes of testicular tissue. Western blot results showed that Wuzi Yanzong recipe significantly increased the expression levels of endoplasmic reticulum stress-related proteins GRP78, p-PERK, p-eif2α, ATF4, p-IRE1, XBP1, ATF6 and significantly decreased the expression levels of endoplasmic reticulum-induced apoptosis-related proteins CHOP, caspase 12 and p-JNK. In conclusion, Wuzi Yanzong recipe can alleviate the ageing-related apoptosis of testicular germ cells in natural ageing rats by regulating endoplasmic reticulum stress.

[Protective effect of Wuzi Yanzong recipe on testicular DNA oxidative damage in natural ageing rats].

To study the protective effects of Wuzi Yanzong recipe on DNA oxidative damage of testis germ cells in natural ageing rats based on Nrf2/HO-1 signaling pathway and base excision repair (BER). In the study, 16-month-old SPF grade male SD rats were randomly divided into three groups, namely ageing model group, and low and high-dose Wuzi Yanzong recipe groups (WZ, 1, 4 g·kg⁻¹). In addition, 2-month-old SD rats were used as adult control group (10 rats in each group). The ageing model group and the adult control group were fed with normal diet for 4 months. WZ groups were given medicated feed for 4 months. After fasting for 12 hours, the rats were put to death. Then, the testes were immediately removed. The vitality of superoxide dismutase (SOD) and malondialdehyde (MDA) content in testis were detected by xanthine oxidase method and thiobarbituric acid (TBA) method. The levels of Nrf2 and 8-OHdG were detected by immunofluorescence. The protein expression levels of Nrf2, HO-1, NQO1, APE1, OGG1 and XRCC1 were detected by Western blot. Compared with the ageing model group, WZ significantly increased the SOD vitality and decreased MDA content of testis. In addition, immunofluorescence results showed that WZ significantly attenuated testicular DNA oxidative damage and improved antioxidant capacity. Such changes were accompanied by the down-regulation of DNA oxidative damage response protein 8-OHdG levels and the up-regulation of Nrf2 levels. Moreover, Western blot results showed that WZ significantly increased the protein expression levels of Nrf2, HO-1 and NQO1 of the testis germ cells, when compared with ageing model group. In parallel, the protein expression levels of APE1, OGG1 and XRCC1 were significantly decreased. In conclusion, WZ improves ageing-related DNA oxidative damage via Nrf2/HO-1 and BER pathways.

[Protective effect of Wuzi Yanzong recipe on testicular DNA damage and apoptosis in natural ageing rats].

To study the protective effect of Wuzi Yanzong recipe on testicular DNA damage and apoptosis in natural ageing rats, SPF grade 16-month-old SD male rats were randomly divided into three groups: ageing model group, low and high dose Wuzi Yanzong recipe groups (WZ, 1， 4 g·kg⁻¹). In addition, 2-month-old SD rats were used as adult control group (10 rats in each group). The ageing model group and the adult control group were fed with normal diet for 4 months. Wuzi Yanzong groups received medicated feed for 4 months. After fasting for 12 hours, the rats were sacrificed. Then testis tissues were taken and weighed to calculate the testis index. The change of testicular tissue morphology was observed by HE staining. Expression and localization of DNA damage-associated protein ATR were observed by immunofluorescence. The expressions of DNA damage-related proteins γ-H2AX, Chk1, p-p53 and apoptosis-related proteins Bcl-2 and Bax in testes were detected by Western blot. The apoptosis of testis tissue in rats was detected by using TUNEL. The results showed that as compared with the youth control group, the protein expression levels of γ-H2AX, Chk1, p-p53 and Bax were significantly increased while Bcl-2 protein expression level was significantly decreased intestis tissues of ageing model group. Wuzi Yanzong recipe significantly decreased protein expression levels of γ-H2AX, Chk1, p-p53 and Bax and increased Bcl-2 protein expression level as well as Bcl-2/Bax ratio. Immunofluorescence results showed that Wuzi Yanzong recipe could significantly decrease the ageing-induced ATR, increase in testis tissues. TUNEL results showed that Wuzi Yanzong recipe could significantly attenuate the germ cell apoptosis in testicular tissues. All the above results suggest that Wuzi Yanzong recipe could protect the germ cell in testicular tissues of natural ageing rates from DNA damage and apoptosis, and the mechanism may be associated with regulating p53 signaling pathway.

[Protective effects of total saponins of Panax japonicas on HepG2 cell apoptosis induced with palmitic acid].

This study aimed to investigate the molecular mechanism and protective effect of total saponins of Panax japonicas (TSPJ) on HepG2 cells apoptosis induced by palmitic acid (PA).The HepG2 cells were cultured in vitro, and divided into five groups: the control group, the model group, the high-dose group (50 mg·L⁻¹), the middle-dose group (25 mg·L⁻¹) and the low-dose group (12.5 mg·L⁻¹).The cells of the five groups were cultured continuously for 24 hours. The cell viability was measured with MTT. HepG2 cells apoptosis was detected by Hoechest staining and Annexin V-FITC/PI staining. The protein expressions of BCL-2, CHOP and TLR4 were measured with western blotting and flow cytometry analysis. The mRNA expressions of TNF-α, IL-1ß, BCL-2, CHOP and GAPDH were measured with RT-PCR. The results suggested that compared with the control group, the number of HepG2 cells of the model group were reduced significantly (P<0.01), while the number of apoptotic HepG2 cells were increased. Compared with the model group, the number of HepG2 cells of the high-dose group and the middle-dose group were increased significantly (P<0.01), whereas the number of apoptotic HepG2 cells were reduced. Compared with the control group, TNF-α, IL-1ß and CHOP mRNA expressions and CHOP and TLR4 protein expressions in the model group were significantly up-regulated (P<0.01), while BCL-2 protein and mRNA expressions in the model group were significantly decreased (P<0.01). Compared with the model group, TNF-α, IL-1ß and CHOP mRNA expressions and CHOP and TLR4 protein expressions in the high-dose group were significantly decreased (P<0.01), while BCL-2 protein and mRNA expressions in the high-dose group were significantly up-regulated (P<0.01).In conclusion, TSPJ can reduce inflammation and apoptosis induced by palmitic acid, with a certain protective effect on liver cells.

[Protective effects of total saponins of Panax japonicas on non-alcoholic fatty liver disease through regulating expression of miR-199-5p].

To research the effection and probable mechanism for the total saponins of Panax japonicas(TPSJ) in mice on non-alcoholic fatty liver disease. Forty SPF male Kunming mice were randomily divided into four group:control group,NAFLD group, low-dose TPSJ treated group,high-dose TPSJ treated group. High-fatty and high-frutose-diet was applied to eatablish NAFLD model，and TPSJ (100 and 200 mg·kg⁻¹) in feeding were given for the TPSJ groups for 4 weeks. To collect the serum with liver and the ALT and TC of serum were monitored after 4 weeks. The hepatic histopathologic structure was observed by haematoxylin-eosin (HE) staining, RT-PCR and RT-qPCR was applied for the detection of miR-199-5p，VEGFa，HGF，c-Met and protein expression level was detected bv laser confocal microscope.Compared with control group, the level of serum ALT and TC in the model group was higher,the liver of the model group showed that hepatocytes display obvious lipid deposition. Then TPSJ treated showed that markedly improved histopathologic changes, decreased fatty deposition. In the meantime,the expression level of miR-199-5p was significantly decreased, thus the expression of HGF and c-Met were significantly increased. TPSJ play a role of prevention on fatty liver, the machanism maybe by blocking miR-199-5p targeted to c-Met signaling pathways in NAFLD.

[Protective effect of total saponins of Panax notoginseng combined with total flavonoids of epimedium on D-galactose-incuced senescence of H9c2 cell].

To investigate the protective effect of Panax notoginseng saponins combined with total flavonoids of epimedium on D-gal-induced senescence of H9c2 cells and explore its underlying mechanisms. The 50 mol•L⁻¹ D-gal was used to induce H9c2 cells senescence. Different concentrations of TPNS, TFE, and TPNS combined with TFE were used for 4 hours for pre-treatment. D-gal was used to stimulate H9c2 cardiac muscle cells for 24 h. Then in order to determine the best combined scheme, MTT was used to detect cell viability. Cell senescence was identified by ß-galactosidase staining. Levels of reactive oxygen species(ROS) was observed by DCFH-DA detection. The changes of mitochondrial membrane potential were identified by JC-1 detection. Protein levels of silentmating type information regulation 2 Homolog-1(SIRT1), peroxisomal proliferator-activated receptor-coactivator 1α(PGC-1α) and silentmating type information regulation 2 Homolog-3(SIRT3) were detected by western blot analysis. The results showed that TPNS(5 mg•L⁻¹) combined with TFE(5 mg•L⁻¹) had significant synergistic effect on H9c2 myocardial cell proliferation(Q=1.154), so 5 mg•L-1TPNS combined with 5 mg•L⁻¹ TFE was determined as the best scheme. The quantity of ß-galactosidase staining and the fluorescence intensity of ROS were apparently decreased in 5 mg•L⁻¹ TPNS combined with 5 mg•L⁻¹ TFE scheme. Meanwhile, it markedly increased the florescence intensity of mitochondrial membrane potential and enhanced the protein expression of SIRT1, PGC-1α and SIRT3. TPNS combined with TFE could protect H9c2 cells from D-gal-induced senescence. The mechanism might be related to adjusting the signal pathways of SIRT1/PGC-1α, SIRT3, adjusting the structure and function of mitochondria and reducing oxidative stress injury.

[Effect of saponins extracted from Panax japonicus on inhibiting cardiomyocyte apoptosis by AMPK/Sirt1/NF-κB signaling pathway in aging rats].

To investigate the effects of saponins extracted from Panax japonicus(SPJ) on cardiomyocyte apoptosis in natural aging rats and explore its underlying mechanisms. SD male rats were randomly divided into four groups: young control group, natural aging group, SPJ low dose group and SPJ high dose group, with 10 rats in each group. The rats in natural aging group, SPJ low and high dose groups were respectively treated with normal saline, SPJ 10 and 60 mg•kg-1•d-1 from the beginning of 18 month-old, 6 days per week for 6 months till 24 month-old. Then the animals were sacrificed. Their myocardial morphology changes were observed by using haematoxylin-eoin(HE) staining; cardiomyocyte apoptosis was tested by using Tunel assays; and the protein expression levels of Bcl-2, Bax, IL-1ß, TNF-α, AMPK, p-AMPK, Sirt1, and Ac-NF-κB p65 in myocardial tissues of rats were detected by Western blot. The results showed that SPJ could effectively improve the arrangement disorder of myocardial fibers, reduce the infiltration of inflammatory cells and inhibit cardiomyocyte apoptosis in natural aging rats. At the same time, SPJ could significantly inhibit the protein expression of Bax, IL-1ß, TNF-α and Ac-NF-κB p65, and increase the expression of Bcl-2, Bcl-2/Bax, p-AMPK/AMPK and Sirt1 in the heart tissues of natural aging rats. SPJ can effectively inhibit cardiomyocyte apoptosis in natural aging rats, and its mechanisms may be related with the regulation of inflammatory reaction by AMPK/Sirt1/NF-κB signaling pathway.

Chikusetsusaponin V attenuates lipopolysaccharide-induced liver injury in mice.

Chikusetsusaponin V (CsV), a saponin from Panax japonicus, has been reported to inhibit inflammatory responses in lipopolysaccharide (LPS)-induced macrophage cells. However, whether CsV could alleviate LPS-induced liver injury in vivo and the potential mechanisms involved remain unclear. In the present study, we investigated the anti-inflammatory effects of CsV on LPS-induced acute liver injury in mice and further explored the potential mechanisms involved. Our results showed that CsV significantly attenuated elevation of alanine transaminase (ALT) and aspartate aminotransferase (AST) levels and improved liver histopathological changes in LPS-induced mice. In addition, CsV decreased serum tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) levels and inhibited mRNA expressions of inducible nitric oxide synthase (iNOS), TNF-α and IL-1ß in LPS challenged mice. Furthermore, CsV inhibited nuclear factor kappa B (NF-κB) activation by downregulating phosphorylated NF-κB, IκB-α, ERK, c-Jun N-terminal kinase (JNK) and p38 levels in the liver tissue, which ultimately decreased nucleus NF-κB protein level. In conclusion, our data suggested that CsV could be a promising drug for preventing LPS challenged liver injury since it attenuated LPS-induced inflammatory responses, partly via inhibiting NF-κB and MAPK signaling pathways.

[Protective Effect of Wuzi Yanzong Prescription on Apoptosis in Germ Cells of Mice Induced by Cyclophosphamide].

Objective: To investigate the protective effect and mechanism of Wuzi Yanzong prescription on apoptosis in germ cell of adult male mice induced by cyclophosphamide( CTX). Methods: Male Balb / C mice were randomly divided into normal control group,model group,the low-,medium- and high- dose of Wuzi Yanzong prescription groups( 100 mg / kg,200 mg / kg and 400 mg / kg),with 10 mice in each group. The mice were injected intraperitoneally with CTX( 200 mg / kg) from 4th day,and gave drug once a week,and executed for 4 weeks. Three doses of Wuzi Yanzong prescription were intragastrically administered every day. For normal control group,the same procedure was performed with intraperitoneal normal saline. Twelve hours after giving CTX at last time, all mice were weighed and sacrificed by cervical dislocation. The testis was immediately dissected and weighed, and then calculated the testis index. The pathological changes of testis were observed by HE staining,the apoptosis of germ cells were detected by TUNEL, the expression of apoptosis-related protein Caspase-3,BAX,BCL-2 in testis were examined by Western blot. Results: Compared with normal control group, the body weight, testis weight,testis index,and the expression of BCL-2 protein levels in testis of model control group were significantly decreased, the expression of BAX,Caspase-3 protein levels and apoptosis in testis of model control group were significantly increased. Wuzi Yanzong prescription significantly increased the body weight,testis weight,testis index,the expression of BCL-2 protein, while decreased the levels of BAX and Caspase-3 protein expression, and then led to the reduction in apoptosis of testis. Conclusion: Wuzi Yanzong prescription can effectively protect the apoptosis of germ cell induced by CTX, and its mechanism may be associated with downregulating protein expression of BAX and Caspase-3,and increasing the protein expression of BCL-2.

[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.

[Protective Effect of Total Saponins of Panax japonicus Against H2O2-Induced Oxidative Stress Damage].

OBJECTIVE: To investigate the protective effects of total saponins of Panax japonicus (TSPJ) on H2O2-induced oxidative stress damage in SH-SY5Y cells, and to explore the underlying mechanism. METHODS: SH-SY5Y cells were incubated with 600 µmol/L H2O2 for 12 h, then treated with various concentrations of TSPJ (0.1, 1, 5 and 20 µg/mL) for 12 h,and then incubated with 600 µmol/ L H2O2 for 12 h. Cell viability was detected by MTT assay. Superoxide dicmutase (SOD) activities and malondialdehyde( MDA) contents were measured by biochemical assay kits. Protein levels of Nrf2,p-ERK, and p-P38 were detected by Western blotting. Levels of NQO1 and GCLC mRNA expression were determined by real-time PCR. RESULTS: Compared with control group, H2O2 stimulated the decrease of cell viability and SOD activities as well as the increase of MDA contents, which were reversed by TSPJ treatment. Furthermore, TSPJ treatment up-regulated not only the decreased protein expressions of Nrf2 and p-ERK but also the decreased mRNA expression of NQO1 and GCLC. CONCLUSION: TSPJ can protect SH-SY5Y cells from H2O2-induced oxidative stress damage. The mechanism may be related to up-regulating the phosphorylation of ERK thereby promoting the Nrf2 nuclear translocation and increasing the mRNA expression of antioxidant genes such as NQO1 and GCLC.

[Protective Effects of Total Saponins from Panax japonicus on H2O2Induced Injury via Mitochondia Pathway in SH-SY5Y Cells].

OBJECTIVE: To investigate the protective mechanism of total saponins from Panax japonicus (SPJ) on H2O2 induced injury in SH-SY5Y cells. METHODS: SH-SY5Y cells were divided into three groups: blank control group, model group (600 µmol/L H2O2) and drug treatment groups. Different concentrations of SPJ (0.1, 1, 5 and 20 µg/mL) were incubated with SH-SY5Y cells for 12 hours prior to exposing to 600 µmol/L H2O2 for another 12 h. Mitochondrial membrane potential (MMP) was detected by JC-1 method. Protein expressions of Sirt1 , PGC-1α, Foxo3a, LC3-II and Beclin1 were detected by Western blotting. RESULTS: Compared to the H2O2 model group, SPJ pretreatment significantly increased MMP level and enhanced the protein expressions of Sirt1, PGC-1α, Foxo3a, LC3-II and Beclin1. CONCLUSION: SPJ exerts protective effect on H2O2 induced SH-SY5Y cell injury through mitochondria pathway.

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.

[Study on protective effect of total saponins of Panax japonicus on LPS-induced RAW264. 7 cell inflammation through NF-kappaB pathway].

OBJECTIVE: To observe the anti-inflammatory effect of total saponins of Panax japonicus on LPS-induced RAW264. 7 macrophages. METHOD: The effect of total saponins of P. japonicus of different concentrations on RAW264. 7 cell viability was determined with the MTT method. The NO kit assay was adopted to detect the NO release of total saponins of P. japonicus to LPS-induced RAW264. 7 cells. The enzyme linked immunosorbent assay (ELISA) was used to detect the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta). The reverse transeriptase-polymerase chain reaction (RT-PCR) was used to determine the expression of inducible nitric oxide synthase (iNOS) ,TNF-alpha,IL-1beta. The protein expression of nuclear transcription factor-kappaB p65 (NF-kappaB p65) was tested by Western blot. RESULT: The safe medication range of total saponins of P. japonicus was less than 80 mg x L(-1). Compared with the LPS model group, total saponins of P. japonicus high, middle and low dose groups (0.1, 1, 10, 40 mg x L(-1)) could significantly reduce the secretion of NO, TNF-alpha, IL-1beta of LPS-induced RAW264. 7 cells, and inhibit the expressions of iNOS, TNF-alpha and IL-1beta mRNA and the protein expression of NF-kappaB p65. CONCLUSION: This study preliminarily proves the protective effect of total saponins of P. japonicus on LPS-induced RAW264.7 macrophages. Its action mechanism may be related to NF-kappaB signal pathway.