ABSTRACT
ObjectiveTo investigate the effect of Yishen Tongluo prescription （YSTLP） on apoptosis of renal tubular epithelial cells and explore the mechanism based on endoplasmic reticulum stress pathway of protein kinase R-like endoplasmic reticulum kinase （PERK）/activating transcription factor 4 （ATF4）/transcription factor C/EBP homologous protein （CHOP）. MethodThe db/db mice were randomly divided into model group， valsartan group （10 mg·kg-1）， and low， middle， high-dose YSTLP groups （1， 2.5， 5 g·kg-1）. Samples were collected after eight weeks of drug intervention. In addition， db/m mice in the same litter served as the control group. Human renal tubular epithelial cells （HK-2） were cultured in vitro and divided into the control group， advanced glycated end-product （AGE） group， and AGE + low， middle， and high-dose YSTLP groups （100， 200， 400 mg·L-1）. TdT-mediated dUTP nick end labeling （TUNEL） staining was used to detect the apoptosis rate of HK-2 cells. Methylthiazolyldiphenyl-tetrazolium bromide （MTT） assay was conducted to detect the viability of HK-2 cells. Calcium fluorescence probe staining and luciferase reporter gene method were adopted to detect the luciferase activity of folded protein response element （UPRE） and endoplasmic reticulum stress. Immunohistochemical （IHC） analysis was carried out to measure the protein expressions of phosphorylated PKR （p-PERK）， CHOP， and ATF4. Real-time polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression levels of CHOP and X-box binding protein 1 （XBP1） in mouse kidney and HK-2 cells. Western blot was used to detect the protein expression level of p-PERK， PERK， CHOP， ATF4， B-cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， and cleaved Caspase-3 in mouse kidney and HK-2 cells. ResultIn the cellular assay， HK-2 cell viability was significantly reduced， and the apoptosis rate was elevated in the AGE group compared with the control group （P<0.01）. The mRNA and protein expression levels of apoptosis-related factor Bcl-2 were significantly reduced （P<0.01）， and those of Bax were significantly increased （P<0.01）. The protein expression level of cleaved Caspase-3 was significantly increased （P<0.01）. Compared with the AGE group， YSTLP administration treatment resulted in elevated cell viability and reduced apoptosis rate （P<0.01）. The mRNA and protein expression levels of Bcl-2 were significantly elevated in a time- and dose-dependent manner （P<0.01）， and those of Bax were significantly reduced in a time- and dose-dependent manner. The protein expression level of cleaved Caspase-3 was significantly reduced in a time- and dose-dependent manner （P<0.01）. The intracellular Ca2+ imbalance and UPRE luciferase fluorescence intensity were increased in the AGE group compared with the control group （P<0.01）. The mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 were significantly increased （P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the AGE group， YSTLP effectively improved intracellular Ca2+ imbalance in HK-2 cells and decreased UPRE luciferase fluorescence intensity in a dose-dependent manner （P<0.01）. It reduced the mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 （P<0.01） and the protein expression levels of intracellular p-PERK， CHOP， and ATF4 in a dose- and time-dependent manner （P<0.01）. In animal experiments， the protein expression level of Bcl-2 was significantly reduced（P<0.01）， and that of cleaved Caspase-3 and Bax was significantly increased in the model group compared with the control group （P<0.05）. The protein expression level of Bcl-2 was dose-dependently elevated， and that of cleaved Caspase-3 and Bax was dose-dependently decreased in the YSTLP groups compared with the model group （P<0.01）. Compared with the control group， the mRNA expression levels of CHOP and XBP1 were significantly elevated in the model group （P<0.05， P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the model group， YSTLP significantly decreased the mRNA expression levels of CHOP and XBP1 （P<0.01） and the protein expression levels of p-PERK， CHOP， and ATF4 （P<0.01）. ConclusionYSTLP can effectively inhibit endoplasmic reticulum stress and improve apoptosis of renal tubular epithelial cells， and its mechanism may be related to the regulation of the PERK/AFT4/CHOP pathway.
ABSTRACT
Objective:To investigate the effect of Ziziphi Spinosae Semen （ZSS） and Albiziae Flos （AF） on behavior and endoplasmic reticulum stress endoplasmic reticulum stress protein kinase R-like endoplasmic reticulum kinase （PERK）/activated transcription factor 4 （ATF4）/CCAAT enhancer binding protein （CHOP） pathway in depression model rats， and to explore its antidepressant mechanism. Method:The male SD rats were divided into normal group， model group， ZSS-AF high dose， middle dose and low dose groups （16， 8， 4 g·kg<sup>-1</sup>） and Venlafaxine group （0.008 g·kg<sup>-1</sup>）， <italic>n</italic>=15 in each group. Except the normal group， the depression model was established in the rats of other 5 groups by the method of chronic unpredictable mild stress （CUMS） combined with isolated feeding. The normal group and model group were given with distilled water by gavage when modeling， while other groups received corresponding drug by intragastric administration for 21 days. Behavior changes of rats in each group were observed by the open field test and sugar water consumption test on 1<sup>th </sup>and 21<sup>th</sup>day of the experiment. The protein expressions of PERK， CHOP， B-cell lymphoma-2 associated X protein （Bax） and cysteine-containing aspartate-specific proteases-3（Caspase-3） were detected by Western blot（WB）， the ultrastructural changes of the hippocampus were observed by transmission electron microscope， the apoptosis of hippocampal neurons was observed by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL） method. Result:Compared with the normal group， the scores of open field test and sugar water consumption rate in model group rats decreased （<italic>P</italic><0.01）. Compared with the model group， the scores of open field test and water consumption rate increased （<italic>P</italic><0.01） in ZSS-AF groups and Venlafaxine group. Transmission electron microscope showed that the changes of neuronal damage in hippocampal were revealed in the model group， whereas those neuronal damages were relieved in ZSS-AF groups and Venlafaxine group. TUNEL method showed that the number of apoptotic neurons in hippocampal increased in the model group （<italic>P</italic><0.01）， but decreased in ZSS-AFgroups and Venlafaxine group （<italic>P</italic><0.01）. WB results showed that as compared with the normal group， protein expressions of PERK， CHOP， Bax and Caspase-3 were up-regulated significantly in the model group （<italic>P</italic><0.01）， whereas those were down-regulated in ZSS-AF groups and Venlafaxine group （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:The antidepressant effect of ZSS-AF herbal pair may be correlated with the regulation of endoplasmic reticulum stress PERK/ATF4/-CHOP pathway.
ABSTRACT
Objective: To explore the possible mechanism of Duhuo Jisheng Tang in relieving knee osteoarthritis based on protein kinase R-like endoplasmic reticulum kinase (PERK)/immunoglobulin-binding protein (Bip) signaling pathway.Method: A model of knee osteoarthritis was established by cold stimulation.Rats were randomly divided into blank group,model group,celecoxib group (0.021 g·kg-1),low,medium and high-dose Duhuo Jisheng Tang groups (8.37,16.72,33.48 g·kg-1).Blank group and model group were given equal volume of physiological saline.The changes of knee joint diameter were recorded.The pathological changes of rat articular cartilage were observed by hematoxylin-eosin (HE) staining.The expressions of tumor necrosis factor-alpha (TNF-α),interleukin-1β(IL-1β) and hyaluronic acid (HA) in serum were detected by enzyme-linked immunosorbent assay (ELISA).The mRNA and protein expression levels of PERK,Bip and cysteinyl as parates pecific protein-9(Caspase-9) in cartilage were detected by Real-time PCR and Western blot.Result: The knee joint redn ess and the joint diameter of celecoxib group and high-dose Duhuo Jisheng Tang group were improved,and the joint diameter was reduced significantly (Pα,IL-1β and HA were increased in model group (PPPα,IL-1β and HA in serum of celecoxib group and high-dose Duhuo Jisheng Tang group were decreased (PPPPConclusion: Duhuo Jisheng Tang can alleviate the symptoms of knee osteoarthritis model rats,and its mechanism may be related to the regulation of PERK/Bip signaling pathway in rat cartilage.