Mechanism of Yishen Tongluo Prescription in Inhibiting Endoplasmic Reticulum Stress and Improving Apoptosis of Renal Tubular Epithelial Cells Based on PERK/ATF4/CHOP / 中国实验方剂学杂志

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.

Clinical Observation on Herb-partitioned Moxibustion on Navel Combined with Yishen Tongluo Prescription in Treatment of Infertility Induced by Idiopathic Asthenozoospermia with Kidney-Yang Deficiency and Collateral Obstruction Syndrome / 中国实验方剂学杂志

ObjectiveTo observe the clinical efficacy of herb-partitioned moxibustion on the navel combined with Yishen Tongluo prescription in the treatment of infertility induced by idiopathic asthenozoospermia （iAZS） with kidney-Yang deficiency and collateral obstruction syndrome and its effect on sperm DNA damage and superoxide dismutase （SOD） in the seminal plasma. MethodsA total of 112 eligible patients who met the inclusion criteria were randomly divided into an observation group （56 cases） and a control group （56 cases）. The patients in the observation group were treated with herb-partitioned moxibustion on the navel combined with Yishen Tongluo prescription，while those in the control group received levocarnitine oral liquid. The primary observation indicators included spouse pregnancy rate，progressive motility （PR），and total sperm motility，and the secondary observation indicators included sperm DNA fragmentation index （DFI），SOD in the seminal plasma， and improvement of TCM syndromes. The treatment cycle was 12 weeks. Before and after treatment，the PR，total sperm motility，sperm DFI，SOD in the seminal plasma， and TCM syndrome scores were recorded. The patients were followed up for 12 weeks and the pregnancy status of spouses within 24 weeks （half a year） was recorded. The clinical efficacy of the two groups was evaluated. ResultThe pregnancy rate of spouses in the observation group was 15.69% （8/51）， higher than 3.85% （2/52） in the control group （χ2=4.118，P<0.05）. The total effective rate of the observation group was 88.24%（45/51）， superior to 69.23% （36/52）in the control group （Z=-3.402，P<0.01）. After treatment， PR， total sperm motility，sperm DFI， SOD in the seminal plasma， and TCM syndromes of the two groups were improved compared with those before treatment （P<0.05）， and the observation group was superior to the control group （P<0.05）. ConclusionHerb-partitioned moxibustion on the navel combined with Yishen Tongluo prescription in the treatment of iAZS-induced infertility patients with kidney-Yang deficiency and collateral obstruction syndrome can increase PR，total sperm motility， and SOD level in the seminal plasma， reduce sperm DFI，improve the TCM symptoms of patients， and improve the pregnancy rate of spouses. The mechanism may be attributed to the fact that this treatment can increase the SOD level in the seminal plasma of patients，enhance the body's antioxidant function，protect sperm from oxidative stress damage，and reduce sperm DFI.

Effect and Mechanism of Yishen Tongluo Prescription on miR-514a-5p/TNFSF15 Signaling Pathway in MN Rats / 中国实验方剂学杂志

ObjectiveTo explore the molecular mechanism of Yishen Tongluo prescription in inhibiting the apoptosis of glomerular podocytes in rats with membranous nephropathy （MN） based on the miR-514a-5p/tumor necrosis factor superfamily member 15 （TNFSF15） signaling pathway. MethodEighty SD rats were pre-immunized and injected with cationized bovine serum albumin （C-BSA） into the tail vein for inducing MN， and the successfully modeled MN rats were randomly divided into the model group， high-， middle-， and low-dose （26.44， 13.22， 6.61 g·kg-1） Yishen Tongluo prescription groups， and benazepril （10 mg·kg-1） group， with 10 rats in each group， and another 20 healthy rats were classified into the normal group. Rats in each group were gavaged with the corresponding drugs， once a day， for four successive weeks. After the administration， the 24-hour urine total protein （UTP） level， serum total cholesterol （TC）， triglyceride （TG）， albumin （ALB）， creatinine （SCr）， and urea nitrogen （BUN） levels were measured. The miR-514a-5p and TNFSF15 mRNA expression levels in the rat kidney tissue were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the expression levels of podocyte marker proteins Nephrin， Podocin， Podocalyxin， Synaptopodin， TNFSF15， and podocyte apoptosis-related proteins B lymphocytoma-2 （Bcl-2）-related X protein （Bax）， Bcl-2-associated death promoter （BAD） protein， and B-cell lymphoma-extra large （Bcl-XL） by immunohistochemistry （IHC）. Western blot was used to detect the expression levels of TNFSF15， Bax， BAD， Bcl-2， and BCL-XL in the rat kidney tissue. The apoptosis rate of rat kidney tissue was measured using the in situ end labeling method （Tunnel）. ResultCompared with the normal group， the level of miR-514a-5p in the kidney tissue was significantly reduced （P<0.05）， and the TNFSF15 mRNA expression was significantly increased （P<0.05）. The expression levels of podocyte marker proteins Nephrin， Podocin， Podocalyxin， and Synaptopodin were down-regulated （P<0.05）. The protein expression levels of TNFSF15， Bax， and BAD were increased （P<0.05）， whereas the Bcl-2 and Bcl-XL protein expression levels were decreased （P<0.05）. The number of apoptotic cells diminished significantly （P<0.05）. Compared with the model group， the level of miR-514a-5p in the kidney tissue was significantly increased （P<0.05）， while the level of TNFSF15 mRNA was significantly decreased （P<0.05）. The expression levels of podocyte marker proteins Nephrin， Podocin， podocalyxin， and Synaptopodin were up-regulated （P<0.05）， whereas the TNFSF15， Bax， and BAD protein expression levels were down-regulated （P<0.05）. Bcl-2 and Bcl-XL protein expression levels rose （P<0.05）. The number of apoptotic cells significantly decreased （P<0.05）. ConclusionYishen Tongluo prescription reduces the apoptosis of rat kidney podocytes and alleviates the kidney injury of MN rats through the miR-514a-5p/TNFSF15 signaling pathway.

Renal Protection of Yishen Tongluo Prescription in Rats with Membranous Nephropathy and Its Influence on AMPK/mTOR/ULK1 Signaling Pathway / 中国实验方剂学杂志

Objective:To observe the effects of Yishen Tongluo prescription （YTP） on autophagy-related proteins in rats with membranous nephropathy （MN） and explore its possible molecular mechanism in protecting the kidney. Method:Twenty of 80 Sprague-Dawley （SD） rats were randomly selected as the normal control， and the rest rats were pre-immunized and injected with cationized bovine serum albumin （C-BSA） through the tail vein to induce MN. The SD rats that were successfully modeled were randomized into the model group， benazepril hydrochloride group （10 mg·kg^-1）， and low- （6.61g·kg^-1）， medium- （13.22 g·kg^-1）， and high-dose （26.44 g·kg^-1） YTP groups， and administered with the corresponding drugs by gavage， once a day， for four consecutive weeks. Then the changes in such quantitative indicators as plasma albumin （ALB）， triglyceride （TG）， total cholesterol （TC）， serum creatinine （SCr）， blood urea nitrogen （BUN）， and 24-hour urinary total protein （UTP） were detected， followed by hematoxylin and eosin （HE） staining， Masson's trichrome staining， and periodic Schiff-methenamine （PASM） staining for observing the pathological changes in kidney under the transmission electron microscope （TEM）. The deposition of immunoglobulin G （IgG） and complement 3 （C3） in the glomerulus was detected by fluorescence immunoassay. The expression levels of autophagy marker proteins Beclin-1， microtubule-associated protein light chain 3Ⅱ （LC3Ⅱ）， and p62 were measured by immunohistochemistry （IHC）， and those of related proteins in the adenosine monophosphate-activated protein kinase / mechanisic target of rapamycin/Unc-51-like kinase 1 （AMPK/mTOR/ULK1） signaling pathway were determined by Western blot assy. Result:Compared with the normal group， the model group exhibited significantly increased UTP （<italic>P</italic><0.01） and serum TG and TC （<italic>P</italic><0.01）， decreased ALB （<italic>P</italic><0.01）， disordered glomerular structure， enlarged volume， thickened basement membrane， vacuolated renal tubules， excessively deposited collagen fibers and fuchsinophilic proteins， extensively fused podocyte foot processes， and diffusely deposited IgG and C3 in glomerular capillary loops. Besides， the expression levels of Beclin-1， LC3II， and phosphorylated AMPK （p-AMPK） decreased （<italic>P</italic><0.01）， while those of p62， phosphorylated mTOR （p-mTOR）， and phosphorylated ULK1 （p-ULK1） increased （<italic>P</italic><0.01）. The comparison with the model group revealed that the TG， TC， and UTP levels in the low-， medium-， and high-dose YTP groups and the benazepril hydrochloride group were reduced to varying degrees （<italic>P</italic><0.05， <italic>P</italic><0.01）， whereas the ALB level was increased （<italic>P</italic><0.01）. There was no statistically significant difference in SCr or BUN level. The pathological damages were alleviated. The expression levels of Beclin-1， LC3Ⅱ， and p-AMPK were up-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）， while those of p62， p-mTOR， and p-ULK1 were down-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:YTP protects the kidney of rats with MN possibly by regulating related proteins in the AMPK/mTOR/ULK1 signaling pathway and activating the autophagy.