Raws li Network Pharmacology Thiab Tsiaj Sim Kev Tshawb Fawb Txog Cov nyhuv ntawm Phenylethanoid Glycoside Ntawm Cistanche Tubulosa Ntawm Diabetic Nephropathy Effect

Abstract: Lub Hom Phiaj Los tshawb txog cov txiaj ntsig ntawmCistanchephenylethanol glycosides(CPhGs) raumob ntshav qab zib raum(DKD) raws li kev siv tshuaj network thiab kev sim tsiaj. Methods UHPLC-QE-MS method tau siv los txheeb xyuas covChemical Cheebtsam ntawm CPhGs. TCMSP thiab Swiss Target Prediction databases tau siv los tshuaj xyuas cov khoom tseem ceeb. OMIM thiab GeneCards databases tau siv los muab cov hom phiaj ntsig txog DKD. Tom qab tau txais cov hom phiaj zoo sib xws, STRING lub vev xaib tau siv los tsim kom muaj kev sib cuam tshuam cov protein. DAVID database tau siv rau GO, KEGG txoj kev tsom xam thiab Cytoscape software tau siv los tsim "CPhGs core component-target-pathway" network. Kaum nas raug xaiv los ua pawg ib txwm, thiab cov nas uas tseem tshuav tau noj nrog cov suab thaj thiab cov rog siab thiab muab ib qho kev txhaj tshuaj intraperitoneal ntawm STZ los tsim kom tau ib tus qauv DKD. Lawv tau muab faib ua pawg qauv, dapagliflozin (1 mg / kg) pawg, thiab CPhGs siab thiab nruab nrab thiab qis (500, 25{{40}}, 125 mg/kg) pawg, 10 tsiaj nyob rau hauv txhua pab pawg, kev cuam tshuam tshuaj rau 6 lub lis piam, cov xwm txheej tau pom, lub cev hnyav tau txiav txim siab thiab kuaj ntshav qabzib txhua lub lim tiam; 24 teev cov zis tau sau tom qab koob tshuaj kawg. Tshawb xyuas 24- teev tag nrho cov zis protein (24-UTP); sau cov ntshav los ntawm lub plab aorta thiab txi nws, cais cov ntshav, thiab kuaj urea nitrogen (BUN), uric acid (UA), tag nrho cov cholesterol (TC), triglyceride (TG), thiab ntshav siab. High-density lipoprotein (HDL) thiab low-density lipoprotein (LDL) ntau ntau, HE staining thiab Masson staining tau siv los soj ntsuam cov kev hloov pauv hauv lub raum cov ntaub so ntswg, ELISA tau siv los kuaj ntshav IL-1 , IL{{17} }, thiab TNF- qib, thiab Western blot tau siv los txheeb xyuas cov ntaub so ntswg raum. JAK2, p-JAK2, STAT3, p STAT3, SOCS3 protein qhia. Cov txiaj ntsig Tag nrho ntawm 699 cov khoom tau kuaj pom los ntawm UHPLC-QE-MS thev naus laus zis. Tom qab kev tshuaj xyuas, 16 lub ntsiab lus tseem ceeb ntawm CPhGs thiab 323 lub hom phiaj cuam tshuam tau txais. CPhGs thiab DKD tau sib koom 99 lub hom phiaj. Lub hom phiaj tseem ceeb yog STAT3, SRC, EGFR, thiab lwm yam. KEGG txoj kev tsom xam tau soj ntsuam 151 txoj kev taw qhia, qhia tias IL-17 txoj kev taw qhia, PI3K / Akt signaling pathway, TNF signaling pathway, thiab lwm yam tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev kho mob ntawm DKD los ntawm CPhGs. CPhGs tuaj yeem ua rau lub cev hnyav ntawm DKD nas (P＜0.01), txo cov dej noj (P＜0.01), tswj cov piam thaj thiab lipid metabolism, txo 24-UTP thiab BUN qib (P＜0.01), thiab txhim kho cov ntaub so ntswg raum. . Pathological kev puas tsuaj txo cov protein qhia ntawm p-JAK2 / JAK2 thiab p-STAT3 / STAT3 hauv lub raum cov ntaub so ntswg (P＜0.01) thiab nce cov protein qhia ntawm SOCS3 (P＜0.01). Xaus CPhGs cuam tshuam hauv DKD los ntawm ntau yam khoom, ntau lub hom phiaj, thiab ntau txoj hauv kev, tej zaum los ntawm inhibiting JAK2 /

STAT3 signaling txoj kev.

Ntsiab lus: Cistanche phenylethanol glycoside; ntshav qab zib nephropathy; UHPLC-QE-MS; network pharmacology; tsiaj sim; JAK2 / STAT3 signaling txoj kev

Nws yuav siv sij hawm ntev npaum li cas rau CISTANCHE ua haujlwm rau cov neeg mob raum?

Mob ntshav qab zib mellitus (DKD), raws li ib qho ntawm cov teeb meem microvascular tseem ceeb tshaj plaws ntawm cov ntshav qab zib, tsis yog qhov tseem ceeb ua raumob raum mobtab sis kuj tseem yog qhov ua rau mob raum kawg (ESRD) thoob ntiaj teb. Yog vim li cas [1-2]. Kev kho mob ntawm DKD feem ntau yog thawj zaug tswj ntshav qab zib, ntshav siab, thiab ntshav lipids, thiab thib ob tiv thaiv lub raum ua haujlwm, zam kev siv tshuaj nephrotoxic, thiab txhim kho txoj kev ua neej [3-4]. Txawm li cas los xij, vim qhov nyuaj pathogenesis ntawm DKD thiab tsis muaj lub hom phiaj tshwj xeeb cuam tshuam, cov kev kho mob tam sim no muaj kev txwv thiab tsis muaj txiaj ntsig. Qhov xwm txheej ntawm qhov kawg ntawm DKD tseem nce ntxiv. Yog li ntawd, nws yog ib qho tseem ceeb heev kom nrhiav tau cov tshuaj zoo.

Cistanche Deserticola Y. C. Ma lubCistanche C.Lub qhuav fleshy stems ntawmCistanche tubulosa (Schenk) Wightnrog cov nplooj scaly [5] tau sau thawj zaug hauv "Shen Nong's Materia Medica" thiab tau teev tseg raws li qib siab. Lawv hu ua "suab puam ginseng" [6]. Lawv qab zib, ntsev, thiab sov. , rov qab mus rau lub raum thiab txoj hnyuv loj meridians, thiab muaj cov teebmeem ntawm nourishing raum yang, replenishing essence thiab ntshav, moistening cov hnyuv, thiab laxative. Kev tshawb fawb qhia tau hais tias phenylethanoid glycosides (CPhGs) yog ib qho ntawm nws cov khoom xyaw tseem ceeb thiab yog lub luag haujlwm rau nws txoj kev loj hlob.

Cov ntaub ntawv hauv paus rau exerting pharmacological teebmeem; Cistanche extract muaj tshuaj tiv thaiv kab mob ntshav qab zib [7-8], tiv thaiv kab mob siab [9], tiv thaiv lub raum [10-11], neuroprotective [12-13] thiab lwm yam teebmeem tshuaj, tab sis tam sim no, CPhGs tsis zoo rau kev kho DKD. Nws cov teebmeem thiab cov txheej txheem tsis meej.

Txoj kev tshawb no tau siv network pharmacology thiab molecular docking txoj kev [14], noj CPhGs raws li cov khoom tshawb fawb, kwv yees thiab tshuaj xyuas nws cov khoom xyaw tseem ceeb thiab lub hom phiaj muaj peev xwm rau kev kho mob ntawm DKD, thiab tom qab ntawd tsim kom muaj kev sib xyaw ntawm streptozocin (STZ)-induced siab. DKD nas qauv pub nrog lipid pub tau siv los ua cov ntsiab lus ntawm kev tshawb fawb vivo, muab kev siv rau kev nthuav dav thiab kev siv cov kev kho mob ntsig txog thiab kev tshawb fawb ntxiv ntawm cov txheej txheem ntawm kev ua.

1 khoom

1.1 Tsiaj

70- cov txiv neej hnub nyoog ib asthiv SD nas nrog SPF qib, lub cev hnyav (240 ± 20) g, tau yuav los ntawm Tsiaj Chaw Tshawb Fawb ntawm Xinjiang Medical University [Siv Tsiaj Tsiaj Ntawv Tso Cai No. SYXK (tshiab) {{4} }], ntawm Cov tsiaj tau hloov kho rau 1 lub lis piam ntawm qhov kub ntawm (22 ± 2) degree, qhov txheeb ze av noo ntawm 50% mus rau 70%, thiab 12 h / 12 h alternating lub teeb thiab tsaus voj voog. Qhov kev sim no tau pom zoo los ntawm Pawg Saib Xyuas Kev Ncaj Ncees Tsiaj ntawm Kev Tshawb Fawb Tsiaj Chaw Kho Mob ntawm Xinjiang Medical University (tus lej lej IACUC-20220127-19).

1.2 Reagents thiab tshuaj

CPhGs (batch number 201610, purity >80%) tau muab los ntawm Xinjiang Hotan Dichen Co., Ltd. Dapagliflozin (batch tooj NB2248) tau yuav los ntawm AstraZeneca, UK. Cov khoom noj qab zib thiab cov rog siab (Cat. No. Boaigang-B1135DM) thiab STZ (Batch No. 2021110307) tau yuav los ntawm Beijing Boai Port Biotechnology Co., Ltd.; cov zis microalbumin (Batch No. 20220611), urea nitrogen (Batch No. 20220626), uric acid (Batch No. 20220613), Tag nrho cov cholesterol (batch tooj 20220615), triglycerides (batch naj npawb 20220626), siab npaum li cas ), thiab low-density lipoprotein (batch tooj 20220615) tau yuav los ntawm Nanjing Jiancheng Bioengineering Institute; interleukin-6 (IL-6, Ntau tus lej JL20896), interleukin-1 (IL-1 , ntau tus lej JL20884), thiab qog necrosis factor- (TNF- , ntau tus lej 13202 ) tau yuav los ntawm Shanghai Jianglai Biotechnology Co., Ltd.; JAK2 luav polyclonal antibody (ntau tus lej 22v9316), p-JAK2 luav polyclonal antibody (ntau tus lej 78g9360), STAT3 luav polyclonal antibody (ntau tus lej 15x8824), p-STAT3 luav polyclonal antibody (ntau tus lej 74m1478), SOCS3 luav polyclonal antibody naj npawb 31d4841) tau tag nrho yuav los ntawm Jiangsu Qianke Biotechnology Research Center Co., Ltd.; HRP-labeled tshis tiv thaiv luav IgG polyclonal antibody (batch tooj 20000758) tau yuav los ntawm Wuhan Mitaka Biotechnology Co., Ltd.; -actin luav monoclonal antibody (batch tooj AC230205001), RIPA lysis buffer (batch naj npawb 20220416), BCA Protein concentration determination kits (batch tooj 20230401) tau muas los ntawm Beijing Solebao Technology Co., Ltd.

1. 3 Instruments

Vanquish ultra-high performance kua chromatography, Q Exactive Focus high-resolution mass spectrometer, Heraeus Fresco17 centrifuge, FC tsis siv neeg microplate nyeem ntawv (Thermo Fisher Scientific, USA); JXFSTPRP-24 grinder (Shanghai Jingxin Technology Co., Ltd.); Mingche D24 UV ntshiab dej ntsuas (Lub teb chaws Yelemees Merck Millipore Company); ACQUITY UPLC BEH C18 kem (1. 7 μm, 2. 1 mm × 100 mm) (American Waters Company); Accu Chek Performa ntshav qabzib ntsuas (Swiss Roche Company); CM1850UV cryostat, DM300 upright fluorescence microscope (Leica Company, Lub teb chaws Yelemees); GL-88B vortex meter (Haimen Qilin Bell Instrument Manufacturing Co., Ltd.); electronic analytical balance (Bio-Rad Company, USA).

2 txoj kev

2. 1 UHPLC-QE-MS tsom xam

2.1.1 Kev npaj cov tshuaj ntsuam xyuas

Nyhav 100 mg CPhGs, ntxiv 500 μL 80% methanol, vortex rau 30 s, ultrasonicate nyob rau hauv ib tug dej khov da dej rau 1 h, cia sawv ntawm -40 degree rau 1 h, centrifuge ntawm 4 degree , 12 000 r/min rau 15 min, aspirate lub supernatant, 0. Lim los ntawm 22μm microporous membrane thiab khaws cia rau hauv lub tub yees -80 degree.

2.1.2 Chromatographic tej yam kev mob

ACQUITY UPLC BEH C18 kem (1. 7 μm, 2. 1 mm × 100 mm); mobile theem acetonitrile (A) -1% formic acid (B), gradient elution (0 ~ 11 min, 15% ~ 75% A; 11 mus rau 12 min, 75% mus rau 98% A; 12 mus rau 14 min, 98% A; 14 txog 14. 1 min, 98% txog 15% A; Volume flow rate 0. 5 ml / min; sab kub 35 degree; txhaj koob tshuaj 5 μL.

2.1.3 Mass spectrometry tej yam kev mob

puv-scan ddMS2 puv scan hom; sheath gas ntim ntws tus nqi 45 arb; auxiliary gas volume flow rate 15 arb; capillary kub 400 degree; Kev sib tsoo lub zog hauv NCE hom 15% ~ 30% ~ 45% voj voog; Txau hluav taws xob ± 4.0 kV.

2.2 Kev Tshawb Fawb Tshuaj Network

2. 2.1 CPHGs

Kev tshuaj ntsuam xyuas lub hom phiaj: Txheeb cov tshuaj tshuaj ntsuam xyuas los ntawm UHPLC-QE-MS raws li cov ntsiab lus txheeb ze, thiab tshem tawm cov uas nws cov qauv tshuaj tseem tsis tau txiav txim siab. Nkag mus rau InChIKey serial tooj ntawm cov tshuaj ntsuam cov khoom xyaw rau hauv TCMSP database kom tau txais cov tshuaj pharmacokinetic ntawm txhua cov khoom xyaw. Raws li cov xwm txheej ntawm qhov ncauj bioavailability (OB) > 30% thiab tshuaj-zoo li (DL) > 0.18, cov neeg sib tw ua tau raws li qhov yuav tsum tau ua yuav raug tshuaj xyuas. Nkag mus rau SMILES tus lej ntawm txhua qhov khoom hauv Swiss Target Prediction database los kwv yees lub hom phiaj, thiab tau txais CPhGs tshuaj tiv thaiv lub hom phiaj.

2. 2.2 KWD

Kev soj ntsuam kab mob Siv "mob ntshav qab zib nephropathy" raws li cov lus tshawb fawb los tshawb nrhiav cov hom phiaj kab mob hauv OMIM, GeneCards, thiab NCBI databases.

2. 2. 3 Component-target-kab mob network tsim

Lub hom phiaj ntawm CPhGs Cheebtsam thiab kab mob tau txais los ntawm R software mapping, thiab Cytoscape 3.7.2 software tau siv los tsim kom muaj cov phiaj xwm kev sib koom ua ke, uas qhov kev tshuaj xyuas plug-in kuaj pom cov hom phiaj tseem ceeb.

2. 2. 4 Kev tsim cov protein-protein interaction network (PPI)

Ntshuam cov kev sib tshuam lub hom phiaj cov proteins ntawm cov khoom thiab cov kab mob rau hauv STRING database, tsim cov protein-protein interaction network (PPI), tshem tawm cov proteins uas tsis muaj kev sib txuas ntawm lub network, thiab teeb tsa kev ntseeg siab ntawm cov qhab nia qis tshaj ntawm cov protein sib cuam tshuam rau {{1} }.700 ib. Tsim ib lub network PPI, rub tawm nws TSV cov txiaj ntsig tau, coj mus rau hauv Cytoscape 3.7.2 software, thiab siv cov kev tshuaj ntsuam plug-in txhawm rau tshuaj ntsuam cov hom phiaj tseem ceeb.

2. 2. 5 GO, KEGG analysis

Siv cov lus piav qhia ua haujlwm hauv DAVID cov ntaub ntawv los ua kom muaj txiaj ntsig zoo ntawm kev sib tshuam cov hom phiaj ntawm cov kab mob tiv thaiv. Ua ntej teeb tsa cov kev tshuaj ntsuam xyuas, nkag mus rau lub hom phiaj kev sib tshuam hauv Gene List box, xaiv nom-gene-cim raws li lub hom phiaj tus cim, xaiv Gene List raws li cov ntaub ntawv hom (Tsev Npe), thiab xaiv " Homo Sapiens (Species)" raws li qhov lub hom phiaj (Species). Haiv neeg), pib qhov kev tshuaj xyuas tom qab lees paub qhov ntsuas ntsuas, tau txais GO thiab KEGG cov txiaj ntsig kev tshuaj ntsuam xyuas, thiab tom qab ntawd siv ImageGP online teeb duab platform los kos cov duab npuas ntawm KEGG thiab GO cov txiaj ntsig kev tshuaj xyuas. Cytoscape 3.7.2 software tsim cov khoom siv -target-pathway network duab.

2. 2. 6 Molecular docking

Ua ntej rub tawm daim duab 3D ntawm cov khoom muaj nyob hauv SDF hom los ntawm PubChem database, tom qab ntawd rub tawm 3D daim duab duab ntawm cov phiaj xwm kev sib tshuam hauv PDB hom los ntawm US Global Protein Database (PDB), thiab thaum kawg muab tso rau CB-Dock lub website rau molecular docking. Kuaj qhov tseem ceeb docking conformations thiab pom lawv nrog Pymol.

2.3 Tsiaj sim

2.3.1 Kev ua qauv, pab pawg, thiab kev tswj hwm tshuaj

10 nas raug xaiv los ua pawg ib txwm noj thiab noj nrog cov khoom noj zoo tib yam, thiab cov nas uas seem tau noj nrog cov suab thaj thiab cov rog ntau. Tom qab 6 lub lis piam, lawv tau txhaj tshuaj intraperitoneal ib zaug ntawm 30 mg / kg STZ (0. 1 mmol / L STZ yaj hauv pH 4. 5 sodium citrate buffer), thiab 72 teev tom qab, cov ntshav tau coj los ntawm tus Tsov tus tw. txhawm rau kuaj ntshav qabzib ceev (FBG). Yog tias FBG Ntau dua lossis sib npaug ntawm 16. 7 mmol / L, tus qauv ntshav qab zib tau tsim tiav [15]; txuas ntxiv Lawv tau noj nrog cov suab thaj thiab cov rog siab pub rau 2 lub lis piam. Kev tsim qauv DKD tau txiav txim siab ua tiav raws li cov zis muaj txiaj ntsig zoo thiab 24- teev tag nrho cov zis protein (24-UTP) Ntau dua lossis sib npaug li 20 mg. Ntawm lawv, 3 tuag thaum lub sijhawm ua qauv thiab 7 tsis ua tiav. , tag nrho ntawm 50 tau ua tiav. Cov nas uas tau ua qauv zoo tau muab faib ua cov qauv, pawg dapagliflozin (1 mg / kg), thiab CPhGs siab, nruab nrab, thiab qis (500, 250, 125 mg / kg) pawg. Txhua pawg thawj coj tau tswj hwm intragastrically. Cov nas nyob rau hauv ib txwm pab pawg neeg thiab cov qauv pab pawg neeg tau muab cov tshuaj sib xws, thiab cov nas nyob rau hauv ib txwm pab pawg neeg thiab cov qauv pab pawg neeg tau muab tib cov nqi ntawm saline ib txwm gavage, ib hnub twg rau tag nrho 6 lub lis piam. Thaum lub sijhawm tswj hwm, cov nas nyob hauv txhua pab pawg tshwj tsis yog cov pab pawg ib txwm txuas ntxiv tau muab cov rog rog.

2.3.2 Biochemical Indicator Detection

Tom qab 6 lub lis piam ntawm kev cuam tshuam CPhG, 24- teev cov zis tau sau los ntawm cov nas, thiab 24- teev tag nrho cov zis protein (24-UTP) tau ntsuas; cov ntshav tau sau los ntawm cov leeg nrawm nrawm txhawm rau txheeb xyuas FBG qib; ntshav tau sau los ntawm lub plab aorta, 4 degree, 1500 r / min. Centrifuge rau 10 feeb kom cais cov ntshav thiab kuaj ntshav urea nitrogen.

BUN), uric acid (UA), tag nrho cov cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (low-density lipoprotein LDL).

2.3.3 Kev soj ntsuam ntawm pathological morphology ntawm lub raum cov ntaub so ntswg

Cov nas tau muab tshuaj loog nrog sodium pentobarbital thiab txi. Lub raum raug tev tawm, hnyav, kho nrog 4% paraformaldehyde, lub cev qhuav dej, pob tshab, dipped nyob rau hauv ciab, embedded, thiab sectioned rau hematoxylin-eosin (HE) staining.

Tom qab Masson staining, cov ntaub so ntswg morphology tau pom nyob rau hauv lub tshuab tsom, thiab Image-Pro Plus 6.

2. 3. 4 ELISA

Cov txheej txheem tau siv los txheeb xyuas cov qib ntawm cov ntshav inflammatory yam hauv cov nas. Cov theem ntawm IL-6, IL-1 , thiab TNF- tau kuaj pom raws li cov lus qhia ntawm cov khoom siv.

2. 3. 5 Western blot txoj kev los xyuas txog cov protein qhia

Cov ntaub so ntswg raum tau noj, RIPA lysate tau ntxiv kom tshem tawm tag nrho cov protein, thiab qhov concentration tau txiav txim siab los ntawm txoj kev BCA.

Tom qab sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hloov mus rau PVDF membrane, thiab tom qab thaiv, JAK2 (1: 2000), p-JAK2 (1: 1000), STAT3 (1: 2000), p -STAT3 (1: 2000), SOCS3 (1: 2000), -actin (1: 2000) antibodies, incubate ntawm 4 degree thaum hmo ntuj, ntxiv HRP-labeled IgG thib ob antibody (1: 5000), thiab incubate nyob rau hauv chav tsev kub rau 1 h, tsim nrog electroluminescence kit, raug los ntawm protein gel imager, siv Image J software

Quantitative tsom xam ntawm daim. Siv -actin raws li kev siv sab hauv, qhov sib piv ntawm tus nqi grey ntawm lub hom phiaj band thiab cov ntaub ntawv sab hauv yog qhov qhia txog cov protein.

2.4 Kev txheeb cais

Cov ntaub ntawv tau ua tiav los ntawm SPSS 25.0 software, thiab cov ntaub ntawv tau qhia tias yog (x±s). Ib txoj kev tsom xam ntawm qhov sib txawv tau siv rau kev sib piv ntawm pab pawg. P<0.05 indicates that the difference is statistically significant.