Study On The Mechanism Of Renal Fibrosis And Follicle Atresia Induced By Bavachin Based On ER Stress

Background and objectivePsoraleae is a commonly used and important tonic in clinical practice,and it is also an effective traditional Chinese medicine for the treatment of vitiligo.In recent years,there have been clinical reports on the hepatotoxicity of Psoraleae,which has attracted widespread attention.Whether the long-term clinical application of Psoraleae can lead to the accumulation of other tissues and organs and cause potential toxicity has not been clinically evidenced,nor has it been systematically studied.However,in previous studies,it has been reported that the whole extract of Psoralea has nephrotoxicity,reproductive toxicity,phototoxicity,etc.,and the specific toxic monomer components and in-depth mechanisms need to be further studied.Bavachin is an important toxic component in Psoralea,and our laboratory found that bavachin were the main monomer causing endoplasmic reticulum stress in liver cells through high-content screening of important components in Psoralea.This study continued the previous research,took bavachin as the research object,carried out systematic experiments in vitro and in vivo,explored the potential toxic target organs of bavachin and further explored its toxicity mechanism,and provided theoretical basis and scientific basis information for its clinical safety application.Research contentWe used the model organism zebrafish as the research object to study the toxic target organs of bavachin in vivo and in vitro,and identified the toxic target organs represented by kidney and ovary.On this basis,the related mechanisms and intervention targets of nephrotoxicity and reproductive toxicity were discussed from the perspective of endoplasmic reticulum stress.MethodsAccording to the acute toxicity test results of juvenile and adult fish,the LC₅₀and BMDL values were simulated,and the subsequent administration concentration was determined accordingly.The toxicity of juvenile fish was mainly detected by taking pictures and videos at 24h,48h,72h and 120h to observe and count the effects of BV on the heart,liver,notochord and skin,and 120h to detect the voluntary movement.Preliminary analysis of target organ toxicity in adult fish is mainly by observing the pathological results of heart,liver,kidney,ovary,and muscle by HE staining,and using high-performance liquid chromatography to detect drug distribution in heart,liver,kidney,brain,ovary,and muscle.The nephrotoxicity was further confirmed by Masson staining,and the changes of AQP1 and N-Cadherin molecules were detected by in situ hybridization.In vitro verification of HK2 cells after administration of BV to CCK-8 to detect cell viability,to determine the concentration of subsequent administration,Western Blot assay to detect fibrosis,EMT,endoplasmic reticulum stress-related protein expression,calcium ion probe Fluo-4-am staining Calcium overload was detected,Mitosox staining was used to detect reactive oxygen species,and images were taken with a live cell imager.The EMT-related renal fibrosis pathway mediated by endoplasmic reticulum stress mediated by reactive oxygen species was further verified by the inhibitor of endoplasmic reticulum stress 4-PBA,the agonist tunicamycin,and the reactive oxygen species scavenger NAC.Calcium ion overload was used as the end point to screen natural compounds,and the ginsenoside Rb1 obtained from the screening was used to verify the effectiveness of the pathway.According to the OECD standard,the hepatocyte index,the gonadal index,the golden index of reproductive toxicity,was calculated by weighing,and the content of vitellogenin was detected by Elisa method to further confirm the disturbance of endocrine.An in vitro cultured follicle model was established,and the survival rate of trypan blue staining was detected to confirm that the modeling was successful.The survival rate and maturation rate of isolated follicles were detected 4 hours after administration of BV in vitro.Fluo-4-am was used to detect calcium overload,and in situ hybridization was used to detect changes in endoplasmic reticulum stress-related molecules.Furthermore,4-PBA was used to verify the relationship between endoplasmic reticulum stress and follicular atresia,q PCR was used to detect the transcription levels of endoplasmic reticulum stress-related molecules,and BV and Bip molecules were molecularly docked.Results1.The LC₅₀value of zebrafish embryos was 10.89μM,and the mortality rate was significantly correlated with the concentration and time.The 40μM group started to die.Subsequently,0.25,0.5,and 1μM were selected for experiments.BV did not affect the heart rate,heart shape,voluntary movement and other indicators of zebrafish juveniles,but liver degeneration could be seen.The LC₅₀value of zebrafish adult females was6.012μM,and the BMDL value was 2.55μM.Subsequently,1/2,1/4,and 1/8 BMDL were selected as the high,medium and low dose groups,and there was no effect on general indicators such as conditioning factors and voluntary movements.Circumstances have an impact,indicating that the dose can be administered as a follow-up.BV was used to establish an HPLC standard curve,and the trend line equation between the concentration and the response value was obtained as y=0.57168x+1.4866,R=0.9998.The BV ranged from 0.5 ng/m L to 500 ng/m L with a good linear relationship with a minimum limit of quantification of 0.5 ng/m L.After 21days of semi-static administration of BV in zebrafish,it was distributed in all tissues.The order of drug concentration from large to small is kidney>liver>ovary>muscle>brain>heart.After long-term administration,the accumulation of the drug in the kidney was significantly higher than that in the liver,reaching 119.14±49.22 ng/m L.The results of pathological sections showed that high,medium and low doses of BV did not affect the heart and muscle of adult zebrafish females,but at medium and high doses,the arrangement of liver cells was disordered,the nucleoli atrophy,the nucleus-to-cytoplasmic ratio of the kidney was increased,the diameter of the tube was decreased,and the ovary was reduced.The distribution of follicles at all levels was not affected but the number of atresia was significantly increased.2.In BV-treated zebrafish,there was a marked increase in blue staining in the apical and basal cells of renal tubular epithelial cells and in the interstitium,and diffuse expansion,that is,collagen deposition,occurred in the whole sample,indicating that kidney fibers developed in zebrafish after BV treatment.After 21 days of BV administration,the transcription level of renal tubular epithelial marker AQP1 was significantly decreased,but the content of N-cadherin,one of the EMT markers,was significantly increased.BV significantly reduced HK2 cell viability in a dose-dependent manner.Cell viability was found to decrease to 82.10±2.43%at 25μM.BV significantly increased the expression of TGF-β1,Smad 3,α-SMA,Notch1,NICD,c-Myc and Hes1 in a dose-dependent manner.BV dose-dependently upregulated the content of Vimentin,ZEB1 and slug,but decreased the expression of Claudin1 and ZO-1 in HK2 cells.In addition,BV treatment resulted in the loss of specific functional proteins in human renal tubular epithelial cells,including ATP1A1,AQP1,and SLC22A6.The expressions of Bip,p-perk,p-e IF2α,ATF4,IRE1αand CHOP were significantly increased in the BV treatment group in a dose-dependent manner,while the expression of XBP1s was significantly decreased.There were no significant changes in ATF6 content between BV-treated and control groups.Ca²⁺overload was significantly enhanced and increased intracellular ROS.4-PBA significantly reversed BV-induced fibrosis,EMT,elevated expression of endoplasmic reticulum stress-related proteins,calcium overload,and elevated reactive oxygen species,while tunicamycin induced fibrosis similar to BV,EMT,and endoplasmic reticulum stress-related protein expression increased.The reactive oxygen species scavenger NAC and ginsenoside Rb1significantly reversed the BV-induced fibrosis,EMT,increased expression of endoplasmic reticulum stress-related proteins,calcium overload,and increased reactive oxygen species content.3.The hepatic body index,gonadal index and vitellogenin content of zebrafish adult female fish were increased after administration of BV for 21 days.The survival rate of isolated follicles was 89.33%,which confirmed that the modeling was successful.The follicle survival rate and maturation rate decreased in a dependent manner with the increase of BV dose.BV also caused calcium overload and significantly up-regulated the transcription levels of ATF6,XBP1s,IRE1α,ATF4 and PERK.4-PBA significantly reversed the BV-induced decline in follicle survival and maturation,and the transcriptional levels of ATF6,XBP1s,IRE1α,ATF4,and PERK were significantly up-regulated,as well as calcium overload.The BV is molecularly docked with the Bip molecule,and the BV binds to the Bip in the pocket between the two chains of the Bip molecule.The hydroxyl groups of psoralen form hydrogen bonds with amino acid residues on Bip,respectively.ConclusionThis study preliminarily discovered the possible renal toxicity and reproductive toxicity of BV by systematically studying the tissue and organ toxicity of the new model organism zebrafish juvenile and adult fish.Renal toxicity is mainly manifested in renal fibrosis,which is EMT-related renal fibrosis caused by endoplasmic reticulum stress caused by reactive oxygen species,which has been verified in vitro and in vivo.The reproductive toxicity is reflected in the increase of follicular atresia,which was verified by in vitro culture of follicles,and the follicular atresia was caused by endoplasmic reticulum stress.