| ObjectiveThe SD rats were fed with high-fat diet to establish hyperlipidemia and phlegm stasis syndrome animal models as the research object.HuaTan JiangZhuo decoction and its disassembled prescriptions were used to intervene.High-throughput sequencing was used to screen differentially expressed genes and revealed the mechanism of HuaTan JiangZhuo decoction and its disassembled formula in regulating hyperlipidemia and phlegm stasis syndrome.Methods(A)Replication,administration and evaluation of animal models of hyperlipidemia and phlegm stasis syndrome in rats150 male SD rats were randomly divided into normal group(10 rats)and high-fat feeding model group(140 rats).After feeding them with ordinary diet and high-fat diet for 30 days,all the rats were tested for lipids and evaluated if the model was successfully established.Then,the high-fat feeding model group was randomly divided into desease control group,western medicine control group,whole decoction low-medium-high dose group,cangzhu-baizhu low-medium-high dose group,Chenpi-banxia low-medium-high dose group and fulin-zexie low-medium-high dose group.During the experiment,the general conditions such as food intake,water intake,and body weight were recorded.After 4 weeks of administration according to the corresponding dosing regimen,blood was taken for the determination of lipids and the effect of each group after the intervention was evaluated.(B)Study on the Mechanism of "HuaTan JiangZhuo decoction"The liver tissues of 6 rats in each group of normal group,disease group,western medicine group and high-dose group of traditional Chinese medicine were randomly selected,mRNA was extracted,transcriptomic high-throughput sequencing was performed,differentially expressed genes were screened,and the mechanism of HuaTan JiangZhuo decoction and its disassemble prescription on hyperlipidemia and phlegm stasis syndrome was studied Results(A)Hyperlipidemia and phlegm stasis syndrome animal model replicationAfter 30 days of model establishment in 150 male SD rats,the levels of TC and TG in the model group increased,and the HDL-C levels decreased.Compared with the normal control group,the differences were statistically significant(P<0.01).Consistent with the criteria for the disease suggest that successfully replicate the mixed hyperlipidemic animal model.There was no statistically significant difference in serum lipid levels between the control group and each drug group after randomization(P>0.05),suggesting that baseline levels of blood lipids were consistent among groups before drug administration and comparable.After 4 weeks of administration,the TC and LDL-C of the disease control group increased,compared with the normal control group,the difference was statistically significant(P<0.05);the “phlegm stasis syndrome” index of the control group rats : The body weight,liver wet weight,liver index,and Lee’s index(obesity)were all elevated.Compared with the normal control group,the difference was statistically significant(P<0.05).Comprehensive blood lipid levels and "phlegm stasis syndrome" indicators can be considered successfully replicate the hyperlipidemia and phlegm stasis syndrome animal models.(B)Intervention effect of Huatan Jiangzhuo decoction and its disassembled prescriptions1.Lipid-lowering resultsAfter 4 weeks of dosing,rat TC levels: western medicine group,whole decoction group,chenpi-banxia low-dose group,and fulin-zexie high-dose group decreased,and the difference was statistically significant compared with the disease control group(P<0.05);TG levels: cangzhu-baizhu high dose group and fulin-zexie high dose group decreased,compared with the disease control group,the difference was statistically significant(P<0.05);HDL-C levels: cangzhu-baizhu mediuem dose and fulin-zexie high dose group increased,compared with the disease control group,the difference was statistically significant(P<0.05).According to "Assessment of Methods of Assisting Hypolipidemic Function",it was determined that HuaTan JiangZhuo decoction is positive for animal serum cholesterol-lowering test results,fulin-zexie is positive for animal serum lipid-lowering test results,Western medicine and chenpi-banxia is positive for animal serum cholesterol-lowering test results,and cangzhu-baizhu is positive for animal serum triglyceride-lowering test results.2.Other indicators(1)Body weight: cangzhu-baizhu high dose group was reduced,and the difference was statistically significant(P<0.05)compared with the disease control group;(2)Body length: cangzhu-baizhu low and medium dose group,chenpi-banxia low-dose group and whole decoction medium dose group decreased,compared with the disease control group,the difference was statistically significant(P<0.05);(3)liver wet weight: whole decoction high-dose group,chenpi-banxia high dose group decreased,compared with the disease control group,the difference was statistically significant(P<0.05);(4)Hepatic coefficient: whole decoction high-dose group decreased,compared with the disease control group,the difference was statistically significant(P<0.05);(5)Lee’s index: cangzhu-baizhu high dose group decreased,and the difference was statistically significant compared with the disease control group(P<0.05).The above results indicate that HuaTan JiangZhuo decoction and its decomposed prescriptions can control blood lipid levels in rats,especially fulin-zexie.(C)Transcriptome sequencing results1.The RNA extracted from the liver samples of each group and the cDNA library that constructed was up to quality standards.The comparison between Reads and reference genomes obtained by sequencing was highly efficient(73.76%~83.81%).2.Differential genetic screening resultsAccording to the screening index: FC(Fold Change)≥1.5 and FDR(False Discovery Rate)<0.05,the differential genes in comparison of desease control group and the normal control group,1704 were up-regulated,and 798 were down-regulated;Western medicine group has 4 down-regulated;the whole group has 1088 up-regulated and 848 down-regulated;cangzhu-baizhu group has 10 up-regulated and 11 down-regulated;chenpi-banxia group has 103 up-regulated and 275 down-regulated;fulin-zexie group has 434 up-regulated and 776 down-regulated.3.Regulation of glucose metabolism(1)HuaTan JiangZhuo decoction can regulate glucose metabolism by activating Ldha,Insr and inhibiting the expression of Aldh1b1,Gck,Uap1l1,Ldhd,Pklr,Srebf1,Cblc and Abcc8,involving pentose and glucuronate interconversion,galactose Metabolic,ascorbate and aldose metabolism,starch and sucrose metabolism,amino and nucleotide sugar metabolism,pyruvate metabolism,propionic acid metabolism,AMPK signaling pathway,insulin signaling and insulin secretion pathway;(2)Fulin-zexie may inhibit Pfkp,G6 pd,Aldh1b1,Akr1b1,Hk2,Hk3,Nagk,Gnpda1,Pkm,Ldhb,Me2,Ldhd,Pklr,Oxct1,Pik3 cg,Pip4k2a,Pik3 cd,Inpp5d,Synj2,Plcg2,Pik3r5,Scd2,Cblc,Slc2a1,Camk2 d,Prkcb,and Kcnn4,regulates glucose metabolism and involves pentose phosphate pathway,pentose and glucuronate interconversion,fructose and mannose metabolism,galactose metabolism,ascorbate and aldose Metabolism,starch and sucrose metabolism,amino and nucleotide sugar metabolism,pyruvate metabolism,propionic acid metabolism,butyrate metabolism,phosphoinositol metabolism,AMPK signaling pathway,insulin signal pathway and insulin secretion pathway;(3)Chenpi-banxia can regulate glucose metabolism by activating Insr and inhibiting expression of Aldh1b1,Pkm,Me2,Pklr,Pik3 cd,Srebf1,Scd2,and Cblc,involving pentose and glucuronate tautomerism,ascorbate and aldose Metabolism,pyruvate metabolism,phosphoinositol metabolism,AMPK signaling and insulin signaling pathway;(4)Cangzhu-baizhu can regulate glucose metabolism by inhibiting the expression of Aldh1b1,involving pentose and glucuronate transmutation,ascorbate and aldose metabolism,and pyruvate metabolism pathway.(5)Summary of glucose metabolism: Huatan Jiangzhuo Decoction and its disassembled prescriptions all can inhibit the expression of Aldh1b1;In addition,whole decoction,fulin-zexie and chenpi-banxia can inhibit the expression of Pklr and Cblc;whole decoction and Fulin-zexie can inhibit the expression of Ldhd;whole decoction and Chenpi-banxia can activate the expression of Insr and inhibit the expression of Srebf1.4.Lipid metabolism regulation(1)Atorvastatin can regulate lipid metabolism by inhibiting the expression of Pla2g2 d,which involves glycerophospholipid metabolism,ether ester metabolism,arachidonic acid metabolism,linoleic acid metabolism and α-linoleic acid metabolism pathway;(2)HuaTan JiangZhuo decoction can regulate lipid metabolism by activating the expression of Acot2,Acot3,Acox1,Dhcr24,Lss,Cyp3a9,Pnpla7,Ptdss2,Ept1,Cyp4a1,Cyp4b1 and inhibiting the expression of Cyp7a1,Aldh1b1,Chka,and Asah2,involving fatty acid elongation.,fatty acid metabolism,steroid biosynthesis,primary bile acid synthesis,steroid hormone synthesis,glyceride metabolism,glycerophospholipid metabolism,ether ester metabolism,sphingolipid metabolism,arachidonic acid metabolism,linoleic acid metabolism,alpha-linolenic acid Acid metabolism,unsaturated fatty acid synthesis,and PPAR signaling pathway(3)Fulin-zexie may regulates lipid metabolism by activating Acot2,Acot3,Dhcr24,Cyp3a9,Pnpla7,Ept1 and inhibit Ppt1,Ppt2,Scd2,Cyp7a1,Cyp7b1,Aldh1b1,Akr1b1,Pla2g4 a,Lpcat2,Pla2g2 d,Pla2g16,Pld4,Asah2,Hpgds,Ptgs1,Tbxas1,and Olr1,involves fatty acid elongation,fatty acid metabolism,steroid biosynthesis,primary bile acid synthesis,steroid hormone synthesis,glyceride metabolism,glycerophospholipid metabolism,ether ester metabolism,sphingolipid metabolism,Arachidonic acid metabolism,linoleic acid metabolism,α-linoleic acid metabolism,unsaturated fatty acid synthesis,and PPAR signaling pathway(4)Chenpi-banxia could regulate lipid metabolism through the activation of Acox1,Cyp2b1,Ept1,Cyp4b1 and inhibit the expression of Scd2,Cyp7b1,Aldh1b1,Pla2g16,Pla2g7 and Pltp,involving fatty acid metabolism,primary bile acid synthesis,steroid hormone synthesis,Glyceride ester metabolism,glycerophospholipid metabolism,ether ester metabolism,arachidonic acid metabolism,linoleic acid metabolism,α-linoleic acid metabolism,unsaturated fatty acid synthesis,and PPAR signaling pathway;(5)Cangzhu-baizhu can inhibit Aldh1b1 and Pla2g2 d and involves glyceride metabolism,glycerophospholipid metabolism,ether ester metabolism,arachidonic acid metabolism,linoleic acid metabolism,and α-linoleic acid metabolism pathway.(6)Summary of lipid metabolism: Huatan Jiangzhuo Decoction and its disassembled prescriptions all can inhibit the expression of Aldh1b1;In addition,whole prescriptions,Fulin-zexie and Chenpi-banxia can inhibit the expression of Ept1;whole prescriptions and Fulin-zexie can activate the expression of Acot2,Acot3,Dhcr24,Cyp3a9 and Pnpla7 and inhibit the expression of Cyp7a1 and Asah2;Whole prescriptions and Chenpi-banxia can activate the expression of Acox1 and Cyp4b1.5.Inflammatory mediator regulation(1)HuaTan JiangZhuo decoction can reduce the inflammatory response by inhibiting the expression of Cblc and Cdc25 b,involving Jak-STAT signaling pathway and MAPK signaling pathway;(2)Fulin-zexie can reduced the inflammatory response by inhibiting Pim1,Il3 ra,Ifngr2,Il2 rg,Pik3cg,Stat1,Ptpn6,Il21 r,Il10ra,Pik3 cd,Jak3,Il15 ra,Cblc,Pik3r5,Cish,Il2 rb,Csf2ra,Crlf2,Pla2g4 a,Dusp10,Rasgrp1,Ptpn7,Rac2,Prkcb,Dusp5,Stmn1,Fas,Nfkb2,Jund,Map4k1,Tgfb1,Pak1,Relb,Rras,Flna,Birc3,Nfkbia,Lyn,Lck,Ccl4,Syk,Ccl19,Zap70,Cd40,Ccl21,Bcl2a1,Traf1,Tnfaip3,Plcg2 and Btk,and involves Jak-STAT signaling pathway,MAPK signaling pathway and NF-kappa B signaling pathway.(3)Chenpi-banxia can reduce the inflammatory response by inhibiting the expression of Il2 rg,Pik3cd,Cblc,Il2 rb,Csf2ra,Stmn1,Map4k1,Pak1,Lck,Syk,Zap70,Ccl21,Bcl2a1,involving Jak-STAT signaling pathway,MAPK signaling Pathway,and NF-kappa B signaling pathway.(4)Summary of inflammation media: Huatan Jiangzhuo Decoction and Fulin-zexie,Chenpi-banxia can inhibit the expression of Cblc.(D)Q-PCR verification resultsThe key genes that regulate lipid metabolism: CYP7A1,LXRα,PPARγ,and SREBP1-c were validated by Q-PCR.The expression levels of the four genes in HuaTan JiangZhuo decoction group and Fulin-zexie group were reduced,and there was a statistically significant difference compared with the disease control group(P<0.05).The expression level of SREBP1-c gene was decreased in the Cangzhu-baizhu group.Compared with the disease control group,the difference was statistically significant(P<0.05).The verification results are basically consistent with the transcriptome sequencing results.Conclusion(I)SD rats fed with high-fat diet for 30 days can successfully replicate the hyperlipidemia and phlegm stasis syndrome animal models.HuaTan JiangZhuo decoction and its decomposed prescriptions can effectively control hyperlipidemia and phlegm stasis syndrome.The level of blood lipids in rats is comparable to Western medicine.Fulin-zexie has the best effect in reducing blood lipids.(II)Preliminary revealing the mechanism of HuaTan JiangZhuo decoction1.In the regulation of glucose metabolism: Huatan Jiangzhuo Decoction and its disassembled prescriptions all can inhibit the expression of Aldh1b1;In addition,whole decoction,fulin-zexie and chenpi-banxia can inhibit the expression of Pklr and Cblc;whole decoction and Fulin-zexie can inhibit the expression of Ldhd;whole decoction and Chenpi-banxia can activate the expression of Insr and inhibit the expression of Srebf1.2.In the regulation of lipid metabolism: Huatan Jiangzhuo Decoction and its disassembled prescriptions all can inhibit the expression of Aldh1b1;In addition,whole prescriptions,Fulin-zexie and Chenpi-banxia can inhibit the expression of Ept1;whole prescriptions and Fulin-zexie can activate the expression of Acot2,Acot3,Dhcr24,Cyp3a9 and Pnpla7 and inhibit the expression of Cyp7a1 and Asah2;Whole prescriptions and Chenpi-banxia can activate the expression of Acox1 and Cyp4b1.3.In the regulation of inflammatory mediators: Huatan Jiangzhuo Decoction and Fulin-zexie,Chenpi-banxia can inhibit the expression of Cblc. |
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