| BackgroundChronic kidney disease(CKD)has become an increasingly serious global health burden.Cardiovascular disease(CVD)is the leading cause of death in CKD patients.In addition to the involvement of traditional cardiovascular risk factors,CKD-specific non-traditional risk factors,such as uremic toxin accumulation,vascular calcification,inflammation,and oxidative stress,may play a crucial role in the occurrence of cardiovascular events in patients with CKD.Mitochondrial bioenergetic dysfunction is one of the early event in kidney injury,and its duration accelerates the progression of CKD.In CKD models,a significant downregulation of genes related to mitochondrial biogenesis can be observed.In uremic cardiomyopathy,myocardial mitochondria remain in a persistent uncoupled state,resulting in impaired cellular energy reserves and increased susceptibility to metabolic stress in the uremic heart.This suggests that maintaining mitochondrial structure and function is crucial for ensuring energy supply to the heart and kidney and protecting mitochondrial function is essential for preventing and treating CKD-related CVD.The activation of AMP-activated protein kinase(AMPK),a cellular "energy switch," can stimulate mitochondrial biogenesis and mitochondrial DNA replication by directly activating peroxisome proliferator-activated receptor-y coactivator 1α(PGC1α)to protect mitochondrial function.The activity of the AMPK/PGC1α pathway is closely related to mitochondrial function in the context of CKD and CVD.The accumulation of uremic toxins is closely associated with adverse renal and cardiovascular outcomes.Protein-bound uremic toxin indoxyl sulfate(IS)can participate in the pathogenesis of cardiorenal injury by inducing inflammation and damaging the antioxidant systems of cells.Additionally,mitochondria may be the primary target of IS toxicity,which explains its widespread non-specific toxic effects in various organs.Therefore,mitochondrial dysfunction and oxidative stress reactions triggered by impaired AMPK/PGC1α energy metabolism pathway under IS stimulation may be one of the mechanisms underlying the progression of CKD-CVD.Previous studies by our team have shown that the Huan-Shuai Formula,which is based on the principles of nourishing qi and blood,promoting diuresis,and reducing turbidity,can delay the occurrence of kidney endpoints in CKD patients,significantly reduce the levels of circulating uremic toxins indoxyl sulfate and p-cresol sulfate in CKD mice,and improve renal and vascular endothelial pathological injuries.Based on previous studies of traditional Chinese medicine syndrome differentiation and clinical experience,considering that the pathogenesis of CKD combined with CVD is primarily based on renal failure and characterized by dampness,turbidity,stasis,and toxins that lead to stasis,with turbidity and stasis mutually obstructing and damaging the heart and kidneys,we proposed the Jia-Jian-Huan-Shuai Formula that addresses both turbidity and stasis,and treats the heart and kidneys together.The Jia-Jian-Huan-Shuai Formula has demonstrated certain therapeutic effects in improving clinical symptoms,quality of life,and cardiac and renal function indicators in CKD patients with CVD.Uremic toxins share similar pathogenic characteristics with traditional Chinese medicine’s "zhuo du"(turbid toxin).Therefore,we propose the hypothesis that the modified Jia-Jian-Huan-Shuai Formula may improve structural and functional damage to the heart and kidneys induced by IS by activating the AMPK/PGC1α pathway,reducing mitochondrial dysfunction,and restoring the energy homeostasis of the heart and kidneys.To test this hypothesis,we first established a CKD rat model induced by 5/6 nephrectomy and observed the effects of Jia-Jian-Huan-Shuai Formula on the heart and kidney function,fibrosis,AMPK/PGC1α pathway-related molecular expression levels,and mitochondrial energy metabolism-related indicators.We also detected the accumulation of uremic toxins indoxyl sulfate,PCS(p-cresol sulfate),and TMAO(trimethylamine N-oxide)in the circulation/heart and kidney tissues of CKD rats and identified the active ingredients of Jia-Jian-Huan-Shuai Formula using liquid chromatography-mass spectrometry.Next,we used IS to stimulate NRK52E rat renal tubular epithelial cells and H9C2 rat myocardial cells to construct a uremic toxin cell injury model.We observed the effects of Jia-Jian-Huan-Shuai Formula on the viability of heart and kidney cells,fibrosis indicators,AMPK/PGC1α pathway-related molecular expression levels,and mitochondrial energy metabolism-related indicators induced by IS.Our goal is to elucidate the protective effects of Jia-Jian-Huan-Shuai Formula on CKD-CVD and its possible mechanisms from the perspective of energy metabolism,providing a theoretical basis for delaying the progression of CKD-CVD.Objective1.To observe the effects of Jia-Jian-Huan-Shuai Formula on the heart/kidney function,interstitial fibrosis of myocardium/renal tubules,AMPK/PGC1αpathway-related molecular expression levels,and mitochondrial energy metabolism-related indicators in uremic cardiomyopathy rats,as well as its protective effect on IS-induced renal tubular epithelial cell and cardiomyocyte injury and energy status.2.By identifying the effective components,a foundation and direction for subsequent research on individual components of Jia-Jian-Huan-Shuai Formula will be provided.3.To investigate how the Jia-Jian-Huan-Shuai Formula may improve the damage caused to renal tubular epithelial and myocardial cells by IS induction,and enhance their energy status,by exploring the AMPK/PGC1α pathway.4.To observe the effects of Jia-Jian-Huan-Shuai Formula on the accumulation of IS,PCS,and TMAO in circulation/heart-kidney tissues of uremic cardiomyopathy rats.Methods1 In vivo experimentA 5/6 nephrectomy rat model of CKD was established,and the experimental groups were:sham operation group,model group,low/medium/high dose of modified Jia-JianHuan-Shuai Formula group,and losartan potassium group,with a 12-week intervention.The general condition and body weight of rats were recorded,blood pressure was measured,and renal function was evaluated by measuring blood creatinine,blood urea nitrogen,and 24-hour urine protein.Histological evaluation of renal and cardiac tissues was performed using HE staining,MASSON staining,Sirius Red staining,TGFβ1 immunohistochemistry,WGA staining,and TUNEL apoptosis detection to assess inflammation,apoptosis,fibrosis,and hypertrophy of myocardial cells.The expression of TGFβ1 in the heart and kidneys,protein expression of the AMPK/PGC1α pathway,gene expression of PGC1α and SIRT1,as well as absolute quantification of mitochondrial copy numbers in the cardiac and renal tissues of CKD rats were evaluated using western-blot and qRT-PCR techniques.The impact of HSF on the accumulation and distribution of uremic toxins in the heart and kidneys was investigated using AFADESI-MSI(Air flow-assisted desorption electrospray ionization mass spectrometry imaging)technique.2 In vitro experimentsIn vitro experiments were conducted to establish a cell injury model of uremic nephropathy in renal tubular epithelial cells(NRK52E)and cardiac myocytes(H9C2)using IS at a concentration of 1.25mM.Firstly,the effects of different concentrations of IS and Jia-Jian-Huan-Shuai Formula on the viability of NRK52E and H9C2 cells were evaluated using the CCK8 assay.The modeling concentration of IS and the intervention concentration of Jia-Jian-Huan-Shuai Formula were determined.Secondly,the experiments were divided into blank control group,HSF 0.01mg/mL group,and HSF 0.05mg/mL group to investigate the concentration-dependent activation of AMPK by Jia-Jian-Huan-Shuai Formula.The experiments were further divided into 0h,0.5h,6h,and 12h groups to observe the time-dependent activation of AMPK by Jia-JianHuan-Shuai Formula The experiments were then divided into blank control group,IS group,IS+HSF 0.01mg/mL group,and IS+HSF 0.05mg/mL group,and the protein expression of AMPK/PGC1 a pathway,TGFβ1,and αSMA were detected using western blot to investigate the effects of Jia-Jian-Huan-Shuai Formula on the activation of AMPK/PGC1α pathway and anti-fibrosis.Finally,the experiments were divided into blank control group,IS group,HSF group,IS+HSF group,and IS+HSF+Compound C group,and the protein expression of AMPK/PGC1α pathway and TGFβ1 were detected using western blot to investigate the association between Jia-Jian-Huan-Shuai Formula and the AMPK/PGC1α pathway in improving uremic cell injury induced by IS.Mitochondrial ROS staining,mitochondrial membrane potential detection,and celltiterglo bioluminescence detection were used to observe the protective effects of Jia-Jian-Huan-Shuai Formula on the mitochondrial function and cellular energy status of cardiac and renal cells.Results1 In vivo experiments(1)Effects of HSF on general conditions of ratsBody weight:In the 16th week of the experiment,the body weight of the MODEL group rats exhibited a significant decrease compared to the SHAM group(P<0.01);the HSF groups had a slight increase in body weight compared with the CKD group(P<0.05).Blood pressure:Compared with the SHAM group,the MODEL group showed a significant increase in mean arterial pressure(P<0.01);both the HSFs and the control LST were able to effectively lower blood pressure in CKD rats compared with the CKD group(P<0.05).(2)Effects of HSF on renal function of ratsCompared to the SHAM group,the MODEL group of rats exhibited a significant increase in blood creatinine and blood urea nitrogen levels(P<0.001),as well as an increase in 24-hour urinary protein(P<0.01).Treatment with HSF at low,medium,and high doses significantly reduced blood creatinine,blood urea nitrogen,and urinary protein levels in the model rats(P<0.05),with the high-dose group showing the most significant effect(P<0.01).(3)Effects of HSF on histopathological changes in rat kidney tissues with CKDHE Staining:The kidney structure of the SHAM group rats was generally normal.In the MODEL rats,interstitial edema accompanied by significant inflammatory cell infiltration and focal tubular atrophy was observed.Compared with the model group,the HSF and LST groups showed a reduction in inflammatory cell infiltration and alleviation of tubular atrophy.Masson Staining:The fibrosis score in the MODEL group was significantly higher than that in the SHAM group(P<0.001).Compared with the MODEL group,the HSF and LST groups showed a significant decrease in fibrosis score(P<0.01).TGFβ1 Expression:The number of TGFβ1-positive cells in the kidney tissue of MODEL rats was significantly increased compared to the SHAM group(P<0.001).The number of TGFβ1-positive cells in the HSF and LST groups was significantly reduced compared with the MODEL group(P<0.01).TUNEL staining:The results revealed a significant increase in the number of apoptotic cells in the MODEL group compared to the SHAM group,while HSF and LST treatment resulted in a reduction in the number of apoptotic cells compared to the MODEL group.(4)Effects of HSF on TGFβ1 protein expression in the kidney of CKD ratsCompared with the SHAM group,the expression level of TGFβ1 was significantly increased in the MODEL group(P<0.001).Compared with the MODEL group,all treatment groups showed certain therapeutic effects(P<0.05),among which the HH group showed the most significant reduction in TGFβ1 expression in CKD kidney tissue(P<0.01).(5)Effects of HSF on AMPK-related energy metabolism phenotype in the kidney of CKD ratsThe AMPK/PGC1α pathway:In the MODEL group,the activation of AMPK,protein and mRNA expression levels of PGC1α,and mRNA expression level of SIRT1 were significantly inhibited compared to the SHAM group(P<0.01),Compared to the model group,the high-dose HSF group showed a partial recovery in AMPK activation,protein and mRNA expression levels of PGC1α(P<0.05).The medium-dose HSF group exhibited an upregulation in PGC1α protein and mRNA expression levels,as well as SIRT1 mRNA expression level(P<0.05).Renal mitochondrial DNA copy numbers:Significant mtDNA damage was observed in the MODEL group compared to the SHAM group(P<0.01).Treatment with HSF significantly improved the mtDNA damage in the kidneys of the model rats(P<0.01).(6)Effects of HSF on cardiac structure and function in ratsThe HW/BW ratio,HW/TL ratio,and whole heart mass of CKD rats were significantly higher than those of the SHAM group(P<0.001).Compared with the MODEL group,the HM and LST could significantly decrease the HW/BW ratio(P<0.01),and all intervention groups could improve the heart weight increase and HW/TL ratio caused by 5/6 NX-induced CKD in rats(P<0.01).These results suggest that the HSF can improve cardiac hypertrophy in CKD rats.HSF improved cardiac hypertrophy in CKD rats:Compared with the SHAM group,the MODEL group exhibited a significant increase in LVAW;d(P<0.05)and LV mass(P<0.001).Compared with the MODEL group,the HH and LST groups were able to improve left ventricular hypertrophy induced by 5/6 nephrectomy(P<0.05),with the HH group showing a more significant improvement(P<0.01).All intervention groups were able to improve the increase in LV mass induced by 5/6 nephrectomy(P<0.05).HSF improves left ventricular systolic function in CKD rats:Compared with the SHAM group,the left ventricular ejection fraction(LVEF),left ventricular short-axis shortening rate(LVFS),and average global longitudinal strain(GLS)were significantly impaired in the CKD group(P<0.05).In comparison to the MODEL group,each medication intervention group could improve the impairment of LVEF induced by 5/6 NX(P<0.05),and the HM group could improve the reduction of GLS(P<0.05).All concentrations of HSF could alleviate the reduction of LVFS induced by 5/6Nx(P<0.05),and the HH group had the most significant effect(P<0.01).HSF improves left ventricular diastolic function in CKD rats:Compared with the SHAM group,the E/A,E’/A’,IVRT,and RPLSR were all significantly impaired in the MODEL group(P<0.01).In comparison to the MODEL group,the HH and LST groups could improve the decrease of E/A induced by 5/6 NX(P<0.01),and the HM and HH groups could increase the RPLSR of CKD rats to a certain extent(P<0.05).Each medication intervention group could increase the IVRT of CKD rats(P<0.01).Exercise endurance of rats:Compared with the SHAM group,the exercise endurance of CKD rats was severely restricted(P<0.001).In comparison to the MODEL group,the HL and HM groups could significantly improve the exercise endurance of CKD rats(P<0.01).(7)The effects of HSF on pathological changes in rat heart tissueHE staining:There were no apparent pathological changes observed in the myocardium of rats in the SHAM group.Rats in the MODEL group showed myocardial cell hypertrophy,focal inflammatory cell infiltration,and widening of the interstitial space.Compared with the MODEL group,the HSF group and LST group showed a decrease in inflammatory cell infiltration and an improvement in myocardial fiber pathological changes.Sirius Red staining:The interstitial fibrosis area of the myocardium in the CKD rats was significantly increased compared to the SHAM group(P<0.001);both the HSF and LST groups showed a decrease in collagen fiber deposition compared to the MODEL group(P<0.05).TGFβ1 staining:TGFβ1 staining in the myocardium of rats in the SHAM group was mostly negative.The MODEL group showed more TGFβ1-positive cells in the myocardium.Both the HSF and LST groups showed a decrease in TGFβ1-positive cells compared to the MODEL group.WGA staining:The cross-sectional area of myocardial cells in the MODEL group was significantly larger than that of the SHAM group(P<0.01).Compared with the MODEL group,both the HSF and LST treatments significantly reduced the crosssectional area of myocardial cells in CKD rats(P<0.05).TUNEL staining:No significant myocardial cell apoptosis was observed in the SHAM group.The number of apoptotic cells in the MODEL group was significantly increased,while the HSF and LST groups showed a decrease in apoptotic cells compared to the MODEL group.Gene expression of ANP and BNP in rat myocardial tissue:The expression of ANP and BNP genes in CKD rats was significantly higher than that of the SHAM group(ANP,P<0.001;BNP,P<0.01).Compared with the CKD group,the HM and HH,as well as LST treatment,all downregulated ANP and BNP mRNA expression in the myocardium of CKD rats(P<0.05).(8)Effects of HSF on TGFβ1 protein expression in the hearts of CKD rats Compared with the SHAM group,the expression level of TGFβ1 in the MODEL group was significantly increased(P<0.001);compared with the MODEL group,both the HM and LST treatments could decrease TGFβ expression(P<0.05).Among them,HH had the most significant effect in reducing TGFβ1 expression in CKD myocardium(P<0.01).The above results indicate that the uremic cardiomyopathy model has been successfully established.(9)Effects of HSF on AMPK-related energy metabolism phenotype in the hearts of CKD ratsAMPK/PGC1α pathway:In the MODEL group of rats,the activation of AMPK,protein and mRNA expression levels of PGC1α,and mRNA expression level of SIRT1 in the myocardium were significantly suppressed compared to the SHAM group(P<0.01).Compared to the MODEL group,treatment with medium-dose HSF improved the AMPK activity in the myocardium(P<0.05),while high-dose HSF treatment significantly ameliorated the suppression of AMPK,upregulated the protein and mRNA expression of PGC1α,and increased SIRT1 mRNA expression in the myocardium of uremic cardiomyopathy rats(P<0.05).Myocardial mtDNA copy number analysis:Significant mtDNA damage was observed in the myocardium of the MODEL group rats compared to the SHAM group(P<0.001).Treatment with HSF significantly improved the mtDNA damage in the myocardium of the model rats(P<0.01).2 In vitro experiments(1)Determination of drug intervention concentrations for NRK52E cellIntervention concentration of IS:Cell viability decreased significantly at IS concentrations of 1.25 mM and 1.6 mM compared to the Ctrl group(P<0.01).Considering that the concentration of 1.6 mM may have a greater impact on cell viability in subsequent experiments,IS at a concentration of 1.25 mM was selected as the intervention concentration for NRK52E cells.Intervention concentration of HSF:HSF at concentrations of 0.01 mg/mL and 0.05 mg/mL significantly enhanced the cell viability of NRK52E cells compared to the Ctrl group(P<0.05).Therefore,HSF at concentrations of 0.01 mg/mL and 0.05 mg/mL was chosen as the intervention concentration for NRK52E cells in subsequent experiments.Intervention concentration of AMPK inhibitor Compound C:Compound C(CC)at a concentration of 10 μM significantly inhibited the cell viability of NRK52E cells(P<0.05).Considering that CC at a concentration of 2.5 μM had insufficient AMPK inhibitory effects,CC at a concentration of 5 μM was selected as the intervention concentration for NRK52E cells in subsequent experiments.(2)Activation of AMPK in NRK52E cells by HSF in a time-and concentrationdependent mannerCompared to the Ctrl group,all HSF treatments significantly activated AMPK(P<0.05),with HSF 0.05mg/mL showing a more pronounced effect than 0.01mg/mL(P<0.05).The degree of AMPK activation by HSF 0.05mg/mL was statistically different between 6h and 12h treatments compared to immediate treatment(P<0.05 for 6h and P<0.01 for 12h),and AMPK activation was significantly lower at 0.5h and 6h treatments compared to 12h treatment(P<0.01 for 0.5h and P<0.05 for 6h).(3)Activation of the AMPK/PGC1α pathway in NRK52E cells under the IS environment by HSFCompared to the Ctrl group,the IS group showed a significant decrease in pAMPK/AMPK levels and downregulation of PGC1 a protein expression(P<0.001).In comparison to the IS group,the IS+HSF 0.05mg/mL group exhibited a reversal of AMPK inactivation(P<0.01),while both the IS+HSF 0.01mg/mL and IS+HSF 0.05mg/mL groups showed an upregulation of PGC 1α expression(P<0.05).(4)Effects of HSF on NRK52E fibrosis in the IS environmentThe expression levels of TGFβ1 and αSMA proteins were significantly increased in the IS group compared to the Ctrl group(P<0.001).However,the IS+HSF 0.05mg/mL group showed a decrease in TGFβ1 protein levels(P<0.01),and both the IS+HSF 0.01mg/mL and IS+HSF 0.05mg/mL groups exhibited a downregulation of aSMA expression,with the latter showing a more pronounced downregulation trend(P<0.001).(5)Effects of HSF on mitochondria in NRK52E cells in the IS environmentIn comparison to the Ctrl group,the IS group showed an increase in mitochondrial membrane potential loss and ROS production.However,the administration of HSF was able to reverse the mitochondrial membrane potential damage and reduce mitochondrial ROS production.(6)HSF alleviates NRK52E cell injury in the IS environment by activating the AMPK/PGC1α pathwayFibrosis:Compared to the Ctrl group,the IS group showed a significant decrease in pAMPK/AMPK levels and downregulation of PGC1α protein expression(P<0.05),and an upregulation of TGFβ1 expression(P<0.001).Compared to the IS group,the HSF group significantly activated AMPK(P<0.001),upregulated PGC1α expression(P<0.01),and downregulated TGFβ1 protein expression(P<0.01).The IS+HSF group reversed the AMPK inactivation(P<0.05)and PGC1α downregulation(P<0.01)induced by IS in NRK52E cells,and the increase in TGFβ1 expression(P<0.05).Compared to the IS+HSF group,the IS+HF+CC group showed a further decrease in AMPK/PGC1α activation levels(P<0.01)and an increase in TGFβ1 expression(P<0.05).Cellular energy status:Compared to the Ctrl group,the NRK52E cells in the IS group showed a significant decrease in ATP content(P<0.01).Compared to the IS group,the NRK52E cells in the IS+HSF group showed a slight increase in ATP content(P<0.05).However,compared to the IS+HSF group,the NRK52E cells in the IS+HSF+CC group showed a further decrease in ATP content(P<0.01).(7)The drug intervention concentrations for H9C2 cellsThe cell viability significantly decreased when treated with IS at concentrations of 1.25 mM and 1.6 mM and showed statistically significant differences when compared to the control group(P<0.01).At a concentration of 1.6 mM,IS was highly toxic to the cells.Therefore,for subsequent experiments,the concentration of IS used for H9C2 cells was set at 1.25 mM.Regarding HSF intervention,concentrations of 0.01 mg/mL and 0.05 mg/mL were found to promote H9C2 cell proliferation(P<0.05).Therefore,for subsequent experiments,the concentrations of HSF used for NRK52E cells were set at 0.01 mg/mL and 0.05 mg/mL.In terms of the AMPK inhibitor,Compound C,a concentration of 10 μM significantly affected the survival rate of H9C2 cells(P<0.01).For consistency with the second study,a concentration of 5 μM of CC was selected as the intervention concentration for H9C2 cells in subsequent experiments.(8)HSF activates AMPK in a time-dependent manner Compared with the Ctrl group,only HSF 0.05 mg/mL significantly activated AMPK in cardiomyocytes(P<.01);the degree of AMPK activation was statistically different between HSF 0.05 mg/mL administration at 0.5h,6h and 12h compared with the immediate administration(P<0.05),and AMPK activation at 6h and 12h was significantly higher than that in the group with 0.5h administration(6h,P<0.05;12h,P<0.01).(9)HSF activates the AMPK/PGC1α pathway in H9C2 cells under the IS-induced environmentCompared with the Ctrl group,the IS group showed a significant decrease in pAMPK/AMPK and downregulation of PGC1α protein expression(P<0.01);compared with the IS group,AMPK inhibition was restored in both the IS+HSF group(P<0.05),and PGC1α expression was upregulated in both the IS+HSF 0.05 mg/mL group(P<0.01).(10)HSF affects H9C2 fibrosis under the IS-induced environmentCompared with the Ctrl group,the expression levels of TGFβ1 and aSMA proteins were significantly increased in the IS group(TGFβ1,P<0.01;αSMA,P<0.001).Furthermore,compared with the IS group,treatment with HSF in the IS+HSF group was found to decrease the protein level of TGFβ1(P<0.05),while IS+HSF treatment at a concentration of 0.05mg/mL was able to downregulate the expression of aSMA(P<0.01).(11)The effect of HSF on mitochondrial function in H9C2 cells under IS environmentCompared with the Ctrl group,the IS group exhibited a reduction in mitochondrial membrane potential and an increase in mtROS production,whereas the administration of HSF was found to ameliorate the aforementioned conditions in a reversible manner.(12)HSF alleviates H9C2 cell damage under IS environment by activating AMPK/PGC1α pathwayFibrosis:Compared with the Ctrl group,the IS group exhibited a significant decrease in the level of pAMPK/AMPK and protein expression of PGC1α(AMPK,P<0.01;PGC1α,P<0.05),and an upregulation of TGFβ1 expression(P<0.05).In contrast,compared with the IS group,treatment with HSF significantly activated AMPK and upregulated PGC1α expression(P<0.01),while downregulating TGFβ1 protein expression(P<0.01).Furthermore,treatment with HSF in the IS+HSF group reversed IS-induced AMPK inactivation(P<0.05)and TGFβ1 upregulation(P<0.05).Additionally,the IS+HSF+CC group further inhibited PGC1α activity(P<0.05),accompanied by further upregulation of TGFβ1 expression(P<0.01).Moreover,compared with the IS+HSF group,the IS+HSF+CC group further reduced the levels of AMPK/PGC1α activation(AMPK,P<0.01;PGC1α,P<0.05),and led to a further upregulation of TGFβ1 expression(P<0.01).Cellular energy status:Compared with the Ctrl group,the IS intervention group exhibited a significant decrease in the intracellular ATP content of H9C2 cells(P<0.01).In contrast,compared with the IS group,treatment with HSF in the IS+HSF group tended to increase the intracellular ATP content of H9C2 cells.However,compared with the IS+HSF group,the IS+HSF+CC group further reduced the intracellular ATP content of H9C2 cells(P<0.01).3 Determination of active ingredients in Jia-Jian-Huan-Shuai Formula decoction Using HPLC-QTRAP-MS/MS technology,17 active components were identified from the Jia-Jian-Huan-Shuai Formula,including major active ingredients such as gallic acid,emodin,ursolic acid,and berberine.4 Effects of Jia-Jian-Huan-Shuai Formula on circulation and accumulation of uremic toxins IS,PCS,and TMAO in uremic cardiomyopathy rats(1)The effect of HSF on the levels of IS,PCS,and TMAO in the circulation of rats with uremic cardiomyopathyCompared to the SHAM group,rats in the MODEL group showed significant increases in serum concentrations of total/free IS and TMAO(P<0.001)as well as total/free PCS(P<0.01).Treatment with HSF significantly decreased serum levels of TMAO and total/free IS(P<0.001),as well as total/free PCS(Total,P<0.05;free,P<0.01)compared to the MODEL group.(2)Distribution and content of IS,PCS,and TMAO in the kidneys of uremic cardiomyopathy ratsIn the MODEL group,the distribution of IS,PCS,and TMAO in the kidneys of rats was uniform.The content of IS,PCS,and TMAO in the kidneys of rats in the MODEL group was significantly higher than that in the SHAM group(P<0.0001).Compared to the MODEL group,treatment with HSF significantly reduced the content of PCS and TMAO in the kidneys of rats(P<0.0001).The content of IS in the HSF group showed a decreasing trend compared to that in the MODEL group.(3)Distribution and content of IS,PCS,and TMAO in the hearts of uremic cardiomyopathy ratsIn the MODEL group,TMAO was distributed in high density in the epicardium,interventricular septum,and cardiac vascular bundle,while IS and PCS were distributed in high density in the cardiac vascular bundle area.Compared to the SHAM group,rats in the MODEL group showed significantly higher levels of TMAO(P<0.01),IS,and PCS(P<0.0001)in the heart.Treatment with HSF significantly reduced the content of PCS and IS in the heart of rats(P<0.0001),and the content of TMAO was slightly reduced(P<0.05)compared to the MODEL group.ConclusionIn summary,this study confirms the presence of heart-kidney energy metabolism disorders in CKD,with suppressed AMPK activity being a key factor.In vivo experiments demonstrated that the Jia-Jian-Huan-Shuai Formula improved the heart and kidney function and histopathological damage in uremic cardiomyopathy rats.In vitro experiments showed that HSF activated the energy switch AMPK in a time-and/or concentration-dependent manner and exerted a dual effect of maintaining cellular energy homeostasis and alleviating uremic toxin-induced cell damage through the AMPK/PGCla pathway.On the one hand,HSF can enhance the energy metabolism pathway by activating AMPK/PGC1α,upregulating mitochondrial biogenesis,and improving the energy metabolism disorder and fibrosis phenotype of CKD heart and kidney.On the other hand,HSF can reduce the accumulation of uremic toxins in the circulation and heart-kidney tissues of uremic cardiomyopathy rats,promote toxin excretion,and achieve the effects of "treating both the heart and kidney"and "dispelling stasis and detoxification".This study is expected to provide new theoretical basis for exploring the pathogenesis of CKD combined with CVD and new ideas for the prevention and treatment of CKD combined with CVD in both Chinese and Western medicine. |
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