Regulation of Bushen Zhuyun Prescription on Ovarian Endoplasmic Reticulum Stress in Rats with Luteal Phase Defect / 中国实验方剂学杂志

Objective:To investigate the regulatory mechanism of Bushen Zhuyun prescription（BSZYP） on endoplasmic reticulum stress （ERS） in rats with luteal phase defect （LPD） induced by mifepristone. Method:Fifty SD rats were randomly divided into a blank group， a model group， a positive control group （dydrogesterone，0.02 g·kg^-1）， and low-（0.08 g·kg^-1）and high-dose （0.24 g·kg^-1） BSZYP groups. Western blot and Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） were used to detect the mRNA and protein expression levels of immunoglobulin binding protein （BIP）， protein kinase R-like endoplasmic reticulum kinase （PERK）， inositol-requiring enzyme 1 （IRE-1）， activating transcription factor 6 （ATF6）， and C/EBP homologous protein （CHOP）. The enzyme-linked immunosorbent assay （ELISA） was used to detect the serum progesterone （P） and estradiol （E₂） levels. Result:Compared with the blank group， the model group showed the elevated protein expression of BIP， PERK， and CHOP （<italic>P</italic><0.01） and the dwindled mRNA expression of PERK and CHOP （<italic>P</italic><0.05）， while no significant difference was observed in the protein expression of IRE-1 and ATF6， mRNA expression of IRE-1， BIP， and ATF6， and serum E₂ and P levels. Compared with the model group， the positive control group displayed diminished protein expression of CHOP （<italic>P</italic><0.01）， while no significant difference was observed in the protein expression of PERK， IRE-1， BIP， and ATF6， mRNA expression of PERK， IRE-1， BIP， ATF6， and CHOP， and serum levels of E₂ and P. The protein expression of CHOP decreased （<italic>P</italic><0.01） and the mRNA expression of CHOP increased （<italic>P</italic><0.05） in the low-dose BSZYP group， while no significant difference was observed in the mRNA and protein expression of PERK， IRE-1， BIP， and ATF6， and serum E₂ and P levels. In the high-dose BSZYP group， the protein expression of PERK， BIP， and CHOP was down-regulated （<italic>P</italic><0.01）， and the mRNA expression of CHOP was up-regulated （<italic>P</italic><0.01）， while no significant difference was observed in the protein expression of IRE-1 and ATF6， mRNA expression of PERK， IRE-1， BIP， and ATF6， and serum E₂ and P levels. Conclusion:BSZYP can treat LPD by relieving ERS.

Effect of Fuzheng Jiedu Prescription Combined with 5-Fu Against Postoperative Recurrence and Metastasis of Gastric Cancer in Mice： An Exploration Based on Endoplasmic Reticulum Stress / 中国实验方剂学杂志

Objective:To investigate the inhibitory effect of Fuzheng Jiedu prescription（FZJDP） combined with 5-fluorouracil （5-Fu） against postoperative recurrence and metastasis in gastric cancer-bearing mice and explore the possible mechanism based on changes in CD4⁺ T cells， CD8⁺ T cells， and regulatory T （Treg） cells of tumor microenvironment， endoplasmic reticulum stress （ERS） and phosphatidylinositol-3-kinase （PI3K）/protein kinase B（Akt）/mammalian target of rapamycin（mTOR） pathway. Method:Forty 615 mice were randomly divided into the model group，FZJDP （25 g·kg^-1） group，5-Fu（25 mg·kg^-1）group，and combined （25 g·kg^-1 FZJDP + 25 mg·kg^-1 5-Fu）group， with 10 mice in each group. Mouse forestomach carcinoma （MFC） cells were implanted into the inner side of footpad of the left hind paw and the transplanted tumor was then surgically excised to establish a postoperative recurrence model. Hematoxylin-eosin（HE） staining was conducted to observe the pathological changes in mice with gastric cancer recurrence and lung metastasis. The CD4⁺/CD8⁺ T cell ratio in recurrent tumor and the percentages of Treg cells ［CD4⁺，CD25⁺， and forkhead box protein P3 （FOXP3）⁺ cells］ in spleen were detected by flow cytometry. The contents of ERS-related proteins ［78-kDa glucose-regulated protein（GRP78），inositol-requiring enzyme 1 alpha （IRE1<italic>α</italic>），activating transcription factor 6（ATF6），and protein kinase R-like endoplasmic reticulum kinase（PERK）］ and the expression of related proteins in the PI3K/Akt/mTOR signaling pathway were determined by Western blot and immunohistochemistry （IHC）. Result:Compared with the model group， the combined group significantly increased the recurrent inhibition rate （<italic>P</italic><0.05）. The recurrent tumor weight was significantly decreased in each treatment group （<italic>P</italic><0.05）. The number of lung metastases and metastasis rate declined in each treatment group， and the lowest values were observed in the combined group， without any statistical significance. The CD4⁺/CD8⁺ T cell ratios in the FZJDP group and combined group were significantly elevated （<italic>P</italic><0.05）， while the percentages of Treg cells were reduced （<italic>P</italic><0.05）. However， 5-Fu resulted in a significant increase in Treg cell percentage （<italic>P</italic><0.05）. IHC results showed that the protein expression levels of ATF6 （<italic>P</italic><0.05，<italic>P</italic><0.01）， IRE1<italic>α</italic>（<italic>P</italic><0.05），and Akt （<italic>P</italic><0.01） in each treatment group were significantly down-regulated as compared with those in the model group. As revealed by Western blot， the GRP78 expression level in the 5-Fu group was lower than that in the model group （<italic>P</italic><0.05）， and the expression levels of PI3K， phosphorylated Akt （p-Akt）， and mTOR were significantly decreased in the 5-Fu group and the combined group （<italic>P</italic><0.05）. Conclusion:FZJDP combined with 5-Fu reduces postoperative recurrence and metastasis in tumor-bearing mice possibly by inhibiting PI3K/Akt/mTOR signaling pathway， diminishing ERS，and improving tumor immune microenvironment.

Effect and mechanism of Bidens pilosa decoction on non-alcoholic fatty liver induced by high fat and high glucose in mice / 中国中药杂志

This study aimed to investigate the effect and possible mechanism of Bidens pilosa decoction on non-alcoholic fatty liver disease(NAFLD) induced by high fat and high glucose in mice. Bald/c mice were randomly divided into normal group, model group, metformin(200 mg·kg~(-1)) treatment group, Bidens pilosa decoction(10 g·kg~(-1)) treatment group, metformin and B. pilosa decoction(100 mg·kg~(-1)+5 g·kg~(-1)) treatment group. Except for the normal group, mice in the other four groups were fed with high-fat and high-glucose diet for 8 weeks to establish the non-alcoholic fatty liver model. After 4 weeks of treatment, blood was collected from the eyeballs, the mice were sacrificed, and relevant indicators were detected. The results showed that compared with the model group, blood lipid and blood glucose levels of each treatment group were significantly lower(P<0.05); HE staining results showed that liver pathological damage in each treatment group was significantly improved; oil red O staining results showed fat distribution in each treatment group significantly reduced(P<0.01); immunohistochemical staining showed that glucose regulated the protein expression of protein 78(GRP78) in liver tissues of each treatment group was also significantly reduced(P<0.01); Western blot results showed that endoplasmic reticulum stress signal pathway-related factors GRP78, phosphorylated-protein kinase R-like ER kinase(p-PERK), eukaryotic translation-initiation factor 2α(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(Chop), inositol requiring 1α(IRE1α), and cleaved-cysteinyl aspartate specific proteinase 12(cleaved-caspase-12) were significantly reduced(P<0.01). The results of the combined drug treatment group were better than those of the single drug treatment group. These results showed that B. pilosa decoction had the effect in improving non-alcoholic fatty liver, and its mechanism may be related to the down-regulation of the expression of endoplasmic reticulum stress(ERS)-related factors, and the reduction of the apoptosis of hepatocytes caused by ERS and the down-regulation of blood lipid and blood glucose levels.

Investigating Relationship Between Renal Damage and Endoplasmic Reticulum Stress in Diabetic Kidney Disease Rats with Blood Stasis Syndrome Based on Combination of Disease and Syndrome / 中国实验方剂学杂志

Objective:To observe the effect of blood stasis syndrome on renal damage and endoplasmic reticulum stress (ERS) in diabetic kidney disease (DKD) rats, and to explore the relationship between renal syndrome of blood stasis damage and ERS in DKD rats. Method:The 50 Male SD rats of SPF level were selected to establish DKD rat model by high fat and high sugar diet combined with intra-abdominal injection of streptozotocin(STZ). They were randomly divided into normal group, diabetes mellitus group and diabetes mellitus and blood stasis syndrome group（0.05 mg·kg-1）, among which diabetes mellitus and blood stasis syndrome group was prepared by dextran method. The general conditions, hemorheology indexes, 24 h urine protein, serum creatinine and renal pathology of the rats in each group were observed. Immunohistochemical analysis, Western blot and Real-time fluorescent quantitative polymerase chain reaction(Real-time PCR)were used to detect mRNA and protein expressions of endoplasmic reticulum stress-related proteins glucose regulated protein 78(GRP78), activating transcription factor-6(ATF6) and renal fibrosis index fibronectin(FN), and alpha smooth muscle actin(α-SMA). Result:Compared with normal group, the rats in diabetes mellitus group showed polyphagia, polyuria and weight loss, increased hemorheology index of whole blood viscosity and plasma viscosity(P<0.05，P<0.01), increased 24 h urine protein, serum creatinine and urea nitrogen(P<0.01), and increased renal pathology, and increased mRNA and protein expression of GRP78, ATF6, FN and α-SMA(P<0.05，P<0.01). After dextran preparation of blood stasis model. Diabetes mellitus and blood stasis syndrome group increased mortality, signs of change is more obvious in the diabetic group, whole blood viscosity, plasma viscosity, 24 h urine protein ration, serum creatinine and urea nitrogen were significantly higher than those in diabetic rats(P<0.01), pathological changes aggravated in the diabetes group. At the same time， mRNA and protein expressions of GRP78, ATF6, FN, and α-SMA in renal tissue were significantly higher than those in diabetic mellitus group(P<0.05，P<0.01). Conclusion:Under the combined disease and syndrome model, the blood stasis syndrome may further aggravate the pathological damage of the kidney of DKD rats, and is related to the enhancement of ERS in the kidney of DKD rats.

Role of inositol-requiring kinase 1α/X-box binding protein 1 in airway mucus secretion induced by neutrophil elastase / 上海交通大学学报（医学版）

Objective: To explore the effect of endoplasmic reticulum stress (ERS) on neutrophil elastase (NE) induced mucin 5AC (MUC5AC) production in human airway epithelial cells. Methods: HBE16 airway epithelial cells were cultured and pretreated with reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) or ERS inhibitor 4-phenylbutyrate (4-PBA), or transfected with small interfering RNA (siRNA) against inositolrequiring kinase 1α (IRE-1α) or X-box binding protein 1 (XBP-1), respectively before incubation with NE. NE group and blank control group were also set up. ROS production was assayed by detection kit; expression of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like endoplasmic reticulum kinase (pPERK), activating transcription factor 6 (ATF6), phosphorylated IRE-1α (pIRE-1α), and XBP-1 protein was detected by Western blotting; spliced XBP-1 (XBP-1s) mRNA was measured by real-time PCR; levels of MUC5AC protein in culture supernatant and cytoplasm were assayed by ELISA and immunofluorescence. Results: There was an obvious increase of ROS production with strong elevation of GRP78, ATF6, pPERK, and pIRE-1α protein in NE group cells after 24 h, compared with blank control group (P<0.05). The protein and mRNA of XBP-1s, and MUC5AC production also increased obviously (P<0.05). NAC and 4-PBA reduced ERS-related protein expression and MUC5AC production and secretion (P<0.05). Further studies showed that MUC5AC secretion was also blunted by IRE-1α siRNA or XBP-1 siRNA, accompanied with decreased expression of XBP-1s mRNA and protein (P<0.05). Conclusion: NE induces ERS by producing ROS, and increases MUC5AC protein production and secretion; IRE-1α/XBP-1 play a certain role in this process.

Protective effects of total saponins of Panax japonicas on HepG2 cell apoptosis induced with palmitic acid / 中国中药杂志

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 , 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-1β, 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 (<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 (<0.01), whereas the number of apoptotic HepG2 cells were reduced. Compared with the control group, TNF-α, IL-1β and CHOP mRNA expressions and CHOP and TLR4 protein expressions in the model group were significantly up-regulated (<0.01), while BCL-2 protein and mRNA expressions in the model group were significantly decreased (<0.01). Compared with the model group, TNF-α, IL-1β and CHOP mRNA expressions and CHOP and TLR4 protein expressions in the high-dose group were significantly decreased (<0.01), while BCL-2 protein and mRNA expressions in the high-dose group were significantly up-regulated (<0.01).In conclusion, TSPJ can reduce inflammation and apoptosis induced by palmitic acid, with a certain protective effect on liver cells.

Protective effect of earthworm active ingredients against endoplasmic reticulum stress induced L-02 hepatocyte apoptosis / 中国中药杂志

This study focused on the protective effect of earthworm active ingredients (EWAs) on hepatocyte apoptosis induced by endoplasmic reticulum stress (ERS) in L-02 cells. The L-02 cells were cultured in vitro. The cell viability was measured with CCK-8, the apoptosis of L-02 cells was detected by flow cytometry, and the relevant protein and mRNA expressions were detected by Western blot and qPCR. According to the findings, tunicamycin (TM) could obviously reduce the survival rate of L-02 cells in a time-dependent and dose-dependent manner. Compared with normal group, the apoptosis rate in model group was significantly increased (<0.05 or <0.01). The protein and mRNA expressions of ERS-related signal molecules, such as GRP78, PERK, eLF2α, ATF4, CHOP and Bax, were significantly up-regulated (<0.05 or <0.01), while Bcl-2 was significantly down-regulated (<0.05 or <0.01). After the administration with different concentrations of EWAs, compared with model group, EWAs could significantly increase the survival rate ofL-02 hepatocyte and decrease the cell apoptosis rates. It could also reduce the protein and mRNA expressions of ERS-related signal molecules, such as GRP78, PERK, eLF2α, ATF4, CHOP and Bax, in a dose-dependent manner (<0.05 or <0.01) and increased the protein and mRNA expressions of Bcl-2(<0.05 or <0.01). These results showed that EWAs had a significantly protective effect on hepatocyte apoptosis induced by ERS in L-02 cells. Its mechanism may be related to the down-regulation of mRNA and protein expressions of GRP78, PERK, ATF4, eLF2α, CHOP and Bax, and the up-regulation, the relief of ERS and the promotion of the proliferation of impaired L-02 cells.

Effects of resveratrol on expression of renal fibrosis proteins ORP150, GRP78, and GRP94 in rat kidney tissue / 中草药

Objective To assess the effect of resveratrol on the expression of endoplasmic reticulum stress molecular partners150-KD oxygen-regulated protein (ORP150) in renal tissues of rats with unilateral ureteral obstruction (UUO). Methods The UUO rat model of renal interstitial fibrosis was established. The rats were randomly divided into sham operation group, model group, enalapril group, the high-dose, medium-dose and low-dose groups of resveratrol, each group of 10. After 14 d of surgery, rats were sacrificed to collect serum and kidney tissue to detect the level of serum Scratinine (Scr) and blood urea nitrogen (BUN); Pathological changes were stained by HE to evaluate of renal tubular damage index, renal interstitial collagen deposition area was detected by Masson staining, apoptosis of in situ cell in renal tissue was determined by TUNEL assay, and using the western blot for the detection of protein expression of ORP150, GRP78, and GRP94 in renal tissue. Results After comparison of the treatment groups with model group, BUN and Scr levels in serum were significantly reduced in high-dose resveratrol group, the damage degree of renal tubules was significantly reduced, the relative area of the renal interstitial collagen decreased, In addition, the expression of ORP150 was increased (P < 0.05, 0.01), GRP78 and GRP94 proteins expression were significantly reduced, (P < 0.05, 0.01). Moreover, the resveratrol high-dose group seems more effective than enalapril groups in reversing the phenotype (P < 0.05). Conclusion Resveratrol was able to protect renal function, alleviate hydronephrosis, reduce interstitial injury of renal tubular, induce the expression of the key endoplasmic reticulum stress signal molecule ORP150 and the down-regulated molecular partner GRP78 and GRP94, delay the apoptosis of renal tubular epithelial cell of rats after UUO and inhibit renal interstitial fibrosis caused by apoptosis deposition.

Effect of formononetin on protein expression of ASK1 and JNK in rats after unilateral ureteral obstruction / 中草药

Objective To study the effect of formononetin on the expression of ASK1 and JNK on the protein level in rat after unilateral ureteral obstruction (UUO). Methods Rats were then randomly divided into control group, model group, the enalapril group, and the high, medium, and low dose groups of formononetin (100, 50, and 25 mg/kg). Renal interstitial fibrosis (RIF) rats model was established by unilateral ureteral obstruction except the control group. The rats were sacrificed 14 d after surgery, and blood samples were collected to detect serum creatinine (Scr) and blood urea nitrogen (BUN) levels. HE staining was used to observe renal pathologic change and determine renal tubular damage index. The area percentage of RIF was detected by Masson staining. Expressions of ASK1 and JNK protein in kidney were determined by Western blotting. Tubular epithelial cell apoptosis was detected by TUNEL assay. Results Serum Scr, BUN level, tubulointerstitial injury index, RIF, the expressions of ASK1 and JNK protein, and apoptotic index were significantly decreased in the treatment groups when compared with the model group (P < 0.05, 0.01). The high dose group of formononetin was more effective than enalapril group. Conclusion Formononetin inhibited the expressions of ASK1 and JNK protein in UUO rats model. Ultimately renal tubular epithelial cell apoptosis was suppressed and the progression of obstructive nephropathy pathologic process was retarded.