| Chronic kidney disease is characterized by decreased glomerular filtration rate/increased urinary protein excretion,which is irreversible and progressive.Proteinuria is an important marker and risk factor for the progression of chronic kidney disease.Podocytes are wrapped around glomerular filtration barrier capillaries,and the fusion or disappearance of foot processes is an important reason for proteinuria.Podocytes show a high level of basic autophagy,and autophagy disorder is involved in the process of a variety of kidney diseases.Previous studies have shown that Modified Huangqi Chifeng Decoction can reduce urinary protein,reduce autophagosomes in podocytes,down regulate the expression of LC3 and beclin-1,and inhibit excessive autophagy in kidney in rats with adriamycin nephropathy.The bidirectional regulation of autophagy by the autophagy negative regulation signal pathway PI3K/AKT/mTOR and the autophagy positive regulation signal pathway AMPK/mTOR are consistent with the theory of balance of yin and yang and interdependence between yin and yang in traditional Chinese medicine.Based on this understanding,this study further explored the renal protective mechanism of Modified Huangqi Chifeng Decoction on autophagy through the combination of in vivo and in vitro experiments.Part 1 Modified Huangqi Chifeng Decoction regulates autophagy through PI3K/AKT/mTOR and AMPK/mTOR pathways to alleviate renal injury in rats with adriamycin nephropathyObjective:To observe the renal protective effect of Modified Huangqi Chifeng Decoction on rats with adriamycin nephropathy,and to explore the autophagy regulation mechanism of Modified Huangqi Chifeng Decoction based on PI3K/AKT/mTOR and AMPK/mTOR signal pathway.Methods:SD rats were injected with adriamycin(5 mg/kg)through tail vein to establish the model of proteinuria nephropathy.The rats were divided into control group,model group,Modified Huangqi Chifeng Decoction group,3MA group,3MA+Modified Huangqi Chifeng Decoction group and Telmisartan group,and continuous administration for 6 weeks.24 h urinary protein was measured before drug intervention and at the end of the 2nd,4th and 6th weeks after intervention.At the end of the 6th week after administration,the serum biochemical indexes were measured,the pathological changes of kidney were observed by HE,Masson and PAS staining,the ultrastructural changes of kidney were observed by transmission electron microscope,the expression of renal fibrosis indexes FN,LN and Col Ⅳ in renal tissue was analyzed by immunohistochemistry,and the expression of autophagy related proteins and pathway proteins were detected by Western blot and immunofluorescence.Results:(1)24 h urinary protein and biochemical indexes:after 10 days of one-time tail vein injection of adriamycin,the 24 h urinary protein of model rats increased significantly compared with the control group(P<0.01).At the end of the second week after the intervention,the 24 h urinary protein in the Modified Huangqi Chifeng Decoction group,3MA group,3MA+Modified Huangqi Chifeng Decoction group and Telmisartan group decreased,but there was no significant difference compared with the model group.At the end of the 4th week,the 24 h urinary protein in the Modified Huangqi Chifeng Decoction group,3MA+Modified Huangqi Chifeng Decoction group and Telmisartan group decreased significantly,which was statistically different from that in the model group(P<0.05).At the end of the 6th week,compared with the model group,the 24 h urinary protein of the Modified Huangqi Chifeng Decoction group,3MA group,3MA+Modified Huangqi Chifeng Decoction group and Telmisartan group decreased significantly(P<0.05).There was no significant difference in 24 h urinary protein between the Modified Huangqi Chifeng Decoction group,3MA group,3MA+Modified Huangqi Chifeng Decoction group and Telmisartan group(P>0.05).After 6 weeks of drug intervention,blood biochemical indexes were detected.Compared with the control group,urea and serum creatinine in the model group increased(P<0.01).Compared with the model group,Modified Huangqi Chifeng Decoction could reduce urea and serum creatinine(P<0.05),and 3MA could reduce urea(P<0.05),but had no significant effect on serum creatinine(P>0.05).There was no significant difference in urea and serum creatinine among Modified Huangqi Chifeng Decoction group,3MA group and 3MA+Modified Huangqi Chifeng Decoction group(P>0.05).Telmisartan had no significant effect on urea and serum creatinine(P>0.05).Compared with the control group,albumin in the model group decreased(P<0.01);Compared with the model group,Modified Huangqi Chifeng Decoction,3MA and Telmisartan had no significant effect on albumin(P>0.05).Compared with the control group,triglyceride and cholesterol in the model group increased(P<0.01);Compared with the model group,3MA could reduce triglycerides(P<0.05).Modified Huangqi Chifeng Decoction and Telmisartan had no significant effect on triglycerides and cholesterol(P>0.05).(2)Alleviate renal pathological injury:after HE.Masson and PAS staining,the pathological changes of kidney were observed under light microscope.In the kidney tissue of the control group,the glomerular structure was complete,the vascular cavity was clear,the proportion of glomerular capsule was normal,and there was no obvious increase of mesangial cells and deposition of mesangial matrix in the glomerulus;The renal tubules were arranged regularly,and the tubular epithelial cells were normal.In the model group,the glomerulus showed compensatory dilation or atrophy,proliferation of mesangial cells,deposition of mesangial matrix,increase of collagen fibers;The arrangement of renal tubules were disordered,cystic expansion or atrophy,and tubular epithelial cells were swollen and denatured.The glomerular and renal tubular lesions in Modified Huangqi Chifeng Decoction group,3MA group,3MA+Modified Huangqi Chifeng Decoction group and Telmisartan group were reduced to varying degrees.Transmission electron microscopy showed that Modified Huangqi Chifeng Decoction,3MA and Telmisartan could alleviate the foot processes fusion induced by adriamycin.Immunohistochemical results showed that Modified Huangqi Chifeng Decoction,3MA and Telmisartan could down regulate the expression of renal fibrosis indexes FN,LN and Col Ⅳ.(3)Regulate autophagy in the kidney:the expressions of LC3,beclin-1 and p62 in renal tissues of rats in each group were detected by Western blot.Compared with the control group,the expressions of LC3 Ⅱ/LC3 Ⅰ,beclin-1 and p62 in renal tissue of model group were up-regulated(P<0.01),and the expressions of LC3 Ⅱ/LC3 Ⅰ,beclin1 and p62 in Modified Huangqi Chifeng Decoction group,3MA group,3MA+Modified Huangqi Chifeng Decoction group and Telmisartan group were down regulated(P<0.05).The results of LC3 immunofluorescence were consistent with those of Western blot.The expression of LC3 in glomerulus of model group increased,while the expression of LC3 decreased in Modified Huangqi Chifeng Decoction group,3MA group,3MA+Modified Huangqi Chifeng Decoction group and Telmisartan group.(4)Regulate PI3K/AKT/mTOR and AMPK/mTOR autophagy pathways:Western blot was used to detect the expression of p-PI3K,PI3K,p-AKT,AKT,p-AMPK,AMPK,p-mTOR and mTOR in renal tissue of rats in each group.The results showed that in the model group,the expressions of p-PI3K/PI3K,p-AKT,AKT and pmTOR/mTOR decreased(P<0.01),and the expression of p-AMPK/AMPK increased(P<0.01).Compared with the model group,Modified Huangqi Chifeng Decoction could up regulate the expression of p-PI3K/PI3K,p-AKT,AKT and p-mTOR/mTOR(P<0.05),and down regulate the expression of p-AMPK/AMPK(P<0.01);3MA+Modified Huangqi Chifeng Decoction and Telmisartan could up regulate the expressions of p-PI3K/PI3K and p-mTOR/mTOR(P<0.01)and down regulate the expression of p-AMPK/AMPK(P<0.05).PI3K/AKT/mTOR signaling pathway was inhibited and AMPK/mTOR signaling pathway was activated in rats with adriamycin nephropathy,and Modified Huangqi Chifeng Decoction could antagonize this effect.Conclusions:(1)Modified Huangqi Chifeng Decoction can reduce urinary protein and improve renal pathological injury in rats with adriamycin nephropathy.(2)3MA can reduce urinary protein and improve renal pathological injury in rats with adriamycin nephropathy,indicating that excessive autophagy is an important mechanism leading to renal injury.3MA+Modified Huangqi Chifeng Decoction failed to further reduce urinary protein and improve renal pathological injury,indicating that the main mechanism of Modified Huangqi Chifeng Decoction is to inhibit excessive autophagy.(3)Modified Huangqi Chifeng Decoction can protect the kidney by activating PI3K/AKT/mTOR signal pathway and inhibiting AMPK/mTOR signal pathway,inhibiting excessive autophagy.Part 2 Modified Huangqi Chifeng Decoction regulates the autophagy based on the interaction of PI3K/AKT/mTOR and AMPK/mTOR pathway to protect podocytesObjective:By culturing podocytes in vitro,observe the protective effect of Modified Huangqi Chifeng Decoction on podocytes,and explore the autophagy regulation mechanism of Modified Huangqi Chifeng Decoction based on PI3K/AKT/mTOR and AMPK/mTOR signal pathway.Methods:Podocytes were cultured in vitro,and the podocyte injury model induced by adriamycin was established.The activity of podocytes was detected by CCK-8,and the appropriate modeling concentration and time were selected.To prepare the medicated serum of Modified Huangqi Chifeng Decoction.Podocytes were divided into control group,model group,low-dose group of Modified Huangqi Chifeng Decoction,medium-dose group of Modified Huangqi Chifeng Decoction,high-dose group of modified Huangqi Chifeng decoction,3MA group and rapamycin group.The activity of podocytes was detected by CCK-8,the expressions of podocyte injury related proteins nephrin and podocin,and the expressions of autophagy related proteins LC3,beclin-1,p62 and mTOR were detected by Western blot.The autophagosomes in podocytes were observed by transmission electron microscope,and the level of autophagic flux was evaluated by lysosomal fluorescent probe.After the intervention of Pik3r1 siRNA and Prkaal/Prkaa2 overexpression plasmid,the expressions of PI3K,AKT and AMPK were detected by Western blot.Results:(1)Protect podocytes:0.2μg/mL adriamycin was used for 24h to establish the podocyte injury model induced by adriamycin.The activity of podocytes was detected by CCK-8.Compared with the control group,the activity of podocytes in the model group decreased,and the difference was statistically significant(P<0.01);Compared with the model group,the low-,medium-and high-doses of medicated serum of Modified Huangqi Chifeng Decoction could improve the activity of podocytes(P<0.01),and 3MA could also improve the acti vity of podocytes(P<0.05).There was no significant difference between rapamycin and the model group.Western blot showed that compared with the control group,the expression of nephrin and podocin in the model group decreased significantly(P<0.01);Compared with the model group,the medicated serum of Modified Huangqi Chifeng Decoction and 3MA could up regulate the expression of nephrin and podocin,and rapamycin could down regulate the expression of nephrin and podocin(P<0.05).(2)Regulate autophagy of podocytes:Western blot showed that compared with the control group,the expressions of LC3 Ⅱ/LC3 Ⅰ and beclin-1 in the model group were up-regulated(P<0.01),and the expressions of p62 and p-mTOR/mTOR were down-regulated(P<0.01);Compared with the model group,the medicated serum of Modified Huangqi Chifeng Decoction and 3MA could down regulate the expression of LC3 Ⅱ/LC3 Ⅰ and beclin-1,and up regulate the expression of p62 and p-mTOR/mTOR;Rapamycin could up regulate the expression of LC3 Ⅱ/LC3 Ⅰ and beclin-1(P<0.01).The results of LC3 immunofluorescence were consistent with those of Western blot.The expression of LC3 in podocytes increased in the model group,and decreased after the intervention of medicated serum of Modified Huangqi Chifeng Decoction.Transmission electron microscopy showed that the number of autophagosomes in podocytes decreased in the Modified Huangqi Chifeng Decoction group,and the results of lysosome fluorescence probe showed that lysosomal fluorescence decreased and the level of autophagic flux decreased.(3)Regulate the interaction of PI3K/AKT/mTOR and AMPK/mTOR autophagy pathway:Western blot showed that the expression of p-PI3K/PI3K and p-AKT/AKT decreased(P<0.01)and p-AMPK/AMPK increased(P<0.01)after adriamycin intervention;Compared with adriamycin intervention group,Modified Huangqi Chifeng Decoction could up regulate the expression of p-PI3K/PI3K and p-AKT/AKT(P<0.01),and down regulate the expression of p-AMPK/AMPK(P<0.01).The expression of PI3K in podocytes was inhibited by Pik3r1 siRNA,which antagonized the activation of PI3K/AKT/mTOR signal pathway by Modified Huangqi Chifeng Decoction,and Modified Huangqi Chifeng Decoction could still down regulate the expression of p-AMPK/AMPK(P<0.01).The expression of AMPK was increased by Prkaal/Prkaa2 overexpression plasmid,which antagonized the inhibitory effect of AMPK/mTOR signaling pathway by Modified Huangqi Chifeng Decoction,and Modified Huangqi Chifeng Decoction could still up regulate the expression of pPI3K/PI3K and p-AKT/AKT(P<0.01).Conclusions:(1)Modified Huangqi Chifeng Decoction can alleviate adriamycin induced podocyte injury.(2)3MA can also alleviate adriamycin induced podocyte injury,while rapamycin aggravates podocyte injury,indicating that adriamycin induced podocyte autophagy is excessive autophagy.Modified Huangqi Chifeng Decoction can down regulate autophagic flux and inhibit excessive autophagy induced by adriamycin.(3)Modified Huangqi Chifeng Decoction can activate PI3K/AKT/mTOR signal pathway,inhibit AMPK/mTOR signal pathway and regulate the interaction between them to maintain autophagy balance,so as to inhibit excessive autophagy and play a protective role of podocytes.Part 3 Component identification of Modified Huangqi Chifeng DecoctionObjective:To identify the components in the decoction and medicated serum in order to explore the material basis of the renal protective effect and autophagy regulation mechanism of Modified Huangqi Chifeng Decoction.Methods:The decoction and medicated serum of Modified Huangqi Chifeng Decoction were prepared.The components of Modified Huangqi Chifeng Decoction were identified by ultra performance liquid chromatography tandem mass spectrometry.Based on the platform of Center of Pharmaceutical Technology Center of Tsinghua university,the components of Modified Huangqi Chifeng Decoction were identified by comparative analysis of primary mass spectrometry and secondary characteristic fragment ions.Results:The decoction and medicated serum components of Modified Huangqi Chifeng Decoction were identified.57 components were detected in the decoction and 20 components were detected in the medicated serum.It is speculated that the renal protective effect and autophagy regulation mechanism of Modified Huangqi Chifeng Decoction are related to the detected components.Conclusions:According to the results and literature research,Astragaloside Ⅳ,calycosin,calycosin-7-O-β-D-glucoside,formononetin,Astragaloside Ⅱ of Astragali Radix;Paeoniflorin and paeonol of Paeoniae Radix Rubra;Protodioscin,diosgenin,dioscin of Dioscoreae Nipponicae Rhizoma;Asperulosidic acid of Hedyotis Diffusa;Gallic acid of Paeoniae Radix Rubra and Euryales Semen are the material basis of the renal protective effect and autophagy regulation mechanism of Modified Huangqi Chifeng Decoction. |
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