Qhov Teebmeem Ntawm Cistanche Shenqiwan Ntawm Lub Raum Kev Puas Tsuaj, GRP78, Thiab Autophagy-txog Yam Tseem Ceeb hauv Zucker Ntshav Qab Zib Fatty Rats Nrog Hom 2 Mob ntshav qab zib mellitus

tus nqi ntawm ib txwm saline. Cov nas tsuag tau noj tsis tu ncua rau 4 lub lis piam. Cov xwm txheej ntawm cov nas tau pom tsis tu ncua. Cov ntshav qabzib yoo mov (FBG) thiab 24- teev urinary microalbumin (24 h U-mAlb) tau ntsuas. Lub raum cov ntaub so ntswg tau stained thiab morphology tau pom. Lub ultra-microstructure tau pom los ntawm kev xa hluav taws xob hluav taws xob microscopy. Cov qib mRNA ntawm GRP78, Beclin-1, thiab LC3 hauv nas raum cov ntaub so ntswg tau kuaj xyuas los ntawm PCR lub sijhawm tiag tiag thiab cov qib protein tau tshuaj xyuas los ntawm Western blotting.Cov txiaj ntsig Piv nrog rau pawg qauv, nas hauv Shenqiwan pab pawg pom

txhim kho plaub hau gloss, hnyav nce, thiab ua haujlwm ntau dua; ob qho tib si FBG thiab 24 h U-mAlb qib tau txo qis (P < 0.05). Covlub raumntawm nas nyob rau hauv cov qauv pab pawg neeg pomdilated glomerular capillaries ua, irregularly thickened hauv qab daim nyias nyias, widened mesangial cheeb tsam, thiab segmental sclerosis; Lub raum tubular atrophy thiab lub raum interstitial fibrous cov ntaub so ntswg kuj tau pom nyob rau hauv cov ob lub raum. Cov kev hloov pauv pathological no tau txo qis hauv pawg Shenqiwan. Piv nrog rau pawg qauv, ob qho tib si noob thiab protein ntau ntawm GRP78 tau txo qis (P < 0.05) hauv Shenqiwan pab pawg; qib protein ntawm Beclin-1

raug txo qis (P < {{0}}.05); thiab qib protein ntawm LC3 tau nce (P <0.05). Xaus Shenqiwan tuaj yeem txo lub raum kev puas tsuaj hauv ZDF nas thiab ncua sijhawm ntawm kev mob ntshav qab zib hauv lub raum los ntawm kev txo qis endoplasmic reticulum kev ntxhov siab thiab txhim kho autophagy.

 

Lo lus tseem ceeb: Shenqiwan;hom 2 mob ntshav qab zib mellitus; mob ntshav qab zib raum; endoplasmic reticulum kev nyuaj siab;autophagy; GRP 78; Zucker diabetics fatty nas

Nyem qhov no kom paub ntau ntxiv txog Cistanche Cov Khoom Siv rau Kev Kho Mob raum Kab Mob Ntshav Qab Zib

Nug kom paub meej ntxiv:

wallence.suen@wecistanche.com 0015292862950

 

 

Mob raum mob ntshav qab zib(DKD) yog ib qho teeb meem microvascular tseem ceeb ntawmntshav qab zib mellitusthiab ib qho laj thawj tseem ceeb ntawmkab mob raum kawg[1]. DKD yog ib qho tseem ceebmicrovascular teeb meem ntawm ntshav qab zibthiabib qho tseem ceeb ntawm cov kab mob raum kawg[1]. Cov neeg mob nrog DKD tuaj yeem tshwm sim nrog cov proteinuria tsis tu ncua thiab tsis tu ncuaglomerular pom tus nqi[2]. Covpathogenesis ntawm DKDyog qhov nyuaj, thiab cov kev tshawb fawb tsis ntev los no tau pom tias endoplasmic reticulum stress (ERS) hauv lub raum yog qhov ua rau mob raum kawg. (Lub endoplasmic reticulum stress (ERS) thiab autophagy kuj muaj feem xyuam nrog DKD, thiab kev kawm txog lawv cov kab mob yuav yog ib qho tseem ceeb rau kev kho DKD. DKD zoo ib yam li "kev ploj tuag qis dua" hauv qeb ntawm kev nqhis dej hauv cov tshuaj suav.
Cov tsos mob ntawm nqhis dej thiab haus dej ntau dhau thiab tso zis tau sau tseg hauv Jin Kui Yao (Qhov tseem ceeb ntawm Golden Horoscope), thiabLub raum Qi Pillyog thov raws li lub ntsiab kev kho mob mis. Lub Jin Kui Yao (金匮要略) tau sau cov tsos mob ntawm nqhis dej thiab tso zis ntau dhau thiab tau hais txogLub raum Qi Pillraws li lub ntsiab kev kho mob mis. Kev kho mob ntawm DKD nrog TCM tuaj yeem tiv thaiv lub raum ua haujlwm tau zoo thiab muaj kev nyab xeeb thiab txhim khu kev qha [5-6]. Kev kho mob ntawm DKD nrog cov tshuaj suav tuaj yeem tiv thaiv lub raum ua haujlwm tau zoo thiab muaj kev nyab xeeb thiab txhim khu kev qha [5-6]. Kev ua tau zoo ntawm lub raum Qi Pill hauv tonifying lub raum thiab pab Yang tau raug lees paub hauv cov kev tshawb fawb ntsig txog [7]. Peb txoj kev tshawb fawb yav dhau los kuj Peb txoj kev tshawb fawb yav dhau los kuj tau pom tias cov tshuaj rau lub raum qi muaj kev tiv thaiv zoo ntawm cov qauv thiab kev ua haujlwm ntawm lub raum cov ntaub so ntswg hauv cov nas mob ntshav qab zib [8]. Hauv txoj kev tshawb fawb tam sim no, peb tau pom qhov cuam tshuam ntawm lub raum qi ntsiav tshuaj Hauv txoj kev tshawb fawb tam sim no, peb tau pom qhov cuam tshuam ntawm lub raum qi tshuaj rau lub raum cov ntaub so ntswg puas, ERS, thiab autophagy-cuam tshuam yam hauv Zucker mob ntshav qab zib fatty nas (ZDF). Hauv txoj kev tshawb no, peb tau tshawb xyuas cov txheej txheem ua tau ntawm lub raum puas hauv ZDF nas los ntawm kev soj ntsuam cov teebmeem ntawm lub raum qi tshuaj raulub raum puas tsuaj, ERS thiab autophagy-muaj feem cuam tshuam nrog kho los ntawm lub raum qi tshuaj.

1 Cov khoom siv
1.1 Kev sim tsiaj
Nees nkaum 12-lub lis piam hnub nyoog SPF txiv neej ZDF nas (lub cev hnyav 390-410 g, pub 5C08 zoo nas chow rau 4 lub lis piam kom ua tiav induction ntawm hom 2 mob ntshav qab zib) thiab xya 12-lub lim tiam- laus SPF txiv neej Zucker Lean (ZL) nas (lub cev hnyav 350-380 g) tau yuav los ntawm Beijing Viton Lehua Laboratory Tsiaj Technology Co. Txhua tus nas tau nyob hauv qhov chaw thaiv ntawm Lub Tsev Haujlwm Saib Xyuas Kev Kho Mob, Tuam Tshoj-Nyiv Lub Tsev Kho Mob Friendship , daim ntawv tso cai naj npawb: SYXK (Beijing) 2016-0043, thiab lub platform tau muab ib qho kev ywj siab qhov cua nyob rau hauv lub barrier, tuav ib tug 12-h alternation ntawm lub teeb thiab tsaus, chav tsev kub ntawm 23-25 degree , thiab chav txheeb ze av ntawm 50 feem pua ​​-60 feem pua ​​. Txhua tus nas raug tso cai haus thiab pub dawb thaum lub sijhawm pub mis.

1.2 Kev tshuaj xyuas kev ncaj ncees
Cov tsiaj sim tau pom zoo los ntawm Kev Tshawb Fawb Tsiaj Ethics Sub-committee ntawm Pawg Neeg Kawm Txuj Ci ntawm Beijing University of Traditional Chinese Medicine (BUCM) nrog tus lej pom zoo BUCM-4-2021102801-4034.
1.3 Cov tshuaj tseem ceeb thiab cov tshuaj reagents
Cov ntsiav tshuaj (Shu Di Huang, Shan Yu Fei, Shan Yao, Mudan Pi, Fu Ling,Cistanche, Ze Xie, Gunner's Fructus, Gui Zhi) rau decoction ntawmLub raum QiCov ntsiav tshuaj tau yuav los ntawm Beijing Tong Ren Tang Co. Physiological saline, specification: 500 mL, muab los ntawm Shandong Hualu Pharmaceutical Co. Silver Staining Solution (Beijing Regen Biotechnology Co., Ltd., Lot No. DG0090), Khawv koob SYBR Mixture (Jiangsu Kangwei Century Biotechnology Co., Ltd.), mRNA Reverse Transcription Kit (Thermo Fisher Scientific, Inc.), RNA TRIzol Reagent (RNA TRIzol Reagent), RNA TRIzol Reagent (RNA TRIzol Reagent). RNA TRIzol Reagent (Beijing Solabao Technology Co., Ltd.), Rat Urine Microalbumin ELISA Kit (Jiangsu Kote Biotechnology Co., Ltd., Lot No. KT8560-A), GRP78 BiP Antibody, LC3B Antibody thiab Beclin{ {5}} Antibody (Abcam, UK) (batch tooj: ab108613, ab192890, ab207612), luav anti-GAPDH polyclonal antibody (Proteintech, USA), tshis anti-luv IgG(H&L)-HRP (Beijing Baiao Yijie Technology Co. Ltd.).

 

Cov ntshav qabzib ntsuas, cov ntshav kuaj ntshav strips (Shanghai Roche Diagnostic Products Co. Ltd.), low-temperature high-speed centrifuge (Eppendorf, Germany), fluorescence quantitative PCR instrument, ob chav ntsug electrophoresis instrument thiab hloov electrophoresis instrument (Burroughs Life Medical Products Co. Ltd.), gel imager (Protein Simple, USA), lub teeb tsom iav (Leica, Lub teb chaws Yelemees), kis tau tus mob electron microscope (Leica, Lub teb chaws Yelemees), thiab gel imager. (Leica, Lub teb chaws Yelemees), kis tau tus mob electron microscope (Hitachi, Nyiv).

2 Txoj kev
2.1 Kev npaj cov tshuaj sim
Lub decoction ntawmLub raum-Qi ntsiav tshuajtau npaj los ntawm kev hloov cov koob tshuaj raum-Qi hauv Jin Kui Yao [9] raws li qhov qub thiab niaj hnub piv ntawm Han system ntawm metrology hauv thawj tsab ntawm "Study on the Dose and Proportion of Typhoid Prescriptions" [2015 ], thiab koob tshuaj decoction xav tau rau kev sim yog raws li nram no: Shu Di Huang 24 g, Shan Yu Fei 12 g,Cistanche10 g, Shan Yao 12 g, Mudan Pi 9 g, Fu Ling 9 g, Ze Xie 9 g, Gunnera 3 g, thiab Gui Zhi 3 g. Cov tshuaj tau soaked hauv dej distilled rau 2 teev. Tom qab ntawd lub decoction yog concentrated rau 30 feeb thiab lim los ntawm sterile gauze. Decoct cov tshuaj rau 30 min, ces ntxiv cov tshuaj ntxiv thiab decoct rau 1 h. Tom qab qhov ntsuas kub ntawm cov tshuaj txo qis rau hauv chav tsev kub, cov tshuaj yog concentrated thiab lim los ntawm sterile gauze, thiab cov filtrate tau ntim thiab khaws cia ntawm -20 degree . Cov tshuaj yuav tsum tau khaws cia tsis pub dhau 1 lub lis piam, thiab cov tshuaj yuav tsum tau coj mus rau chav sov ua ntej kev tswj hwm.

2.2 Tsim thiab pab pawg ntawm cov qauv tsiaj
ZDF nas raug ntxias nrog 5C08 noj zaub mov muaj roj ntau rau 4 lub lis piam, thiab cov ntshav qabzib tau ntsuas los ntawm kev sau cov ntshav qab zib thaum muaj hnub nyoog 12 lub lis piam. Cov nas nyob rau hauv lub raum qi ntsiav tshuaj tau muab aqueous decoction ntawm lub raum qi ntsiav tshuaj, thiab txhua hnub koob tshuaj ntawm 8.12 g / kg ib nas raug xam raws li cov qauv hauv "Cov qauv kab mob hauv cov tsiaj sim" [10], thiab cov tshuaj concentration. yog 0.812 g / mL, thiab cov nas nyob rau hauv cov qauv thiab cov pab pawg tswj tau muab saline nyob rau hauv cov ntim sib thooj rau 4 lub lis piam.

2.3 General mob thiab yoo mov ntshav qabzib (FBG) kuaj cov nas
Cov xwm txheej dav dav ntawm cov nas tau pom thiab sau tseg txhua hnub, thiab lub cev hnyav tau hnyav tsis tu ncua. Thaum kawg ntawm 12 lub lis piam thiab 16 lub lis piam ntawm hnub nyoog, cov ntshav tau sau los ntawm tus Tsov tus tw ntawm nas tom qab 8 teev ntawm kev yoo mov, thiab FBG tau ntsuas los ntawm Roche cov ntshav qabzib ntsuas thiab kuaj strips. 2.4 Lub 24-h urinary microalbumin (24-h U-mAlb) tau ntsuas thaum kawg ntawm 16 lub lis piam, thiab 24-h U-mAlb concentration tau ntsuas raws li cov lus qhia ntawm LISA kit.

2.5 Kev sau cov ntaub so ntswg raum
Thaum kawg ntawm 16 lub lis piam, tag nrho cov nas yoo mov rau 8 teev. Tom qab tshuaj loog, lub plab kab noj hniav tau qhib thiab ob lub raum raug nthuav tawm, thiab lub peritoneum raug tshem tawm tsis tu ncua thiab tev tawm. Lub raum cortex tau muab cais thiab ib nrab kho nyob rau hauv 4 feem pua ​​paraformaldehyde rau kev npaj ntawm 3 μm paraffin seem; ib nrab txiav rau hauv 1 mm3 cov ntaub so ntswg blocks thiab tsau nyob rau hauv 2.5 feem pua ​​glutaraldehyde rau kev npaj ntawm ultrathin seem. Cov ntaub so ntswg ntxiv tau khaws cia ntawm -80 degree rau Realtime PCR thiab Western blotting assays.

 

2.6 Kev soj ntsuam morphological ntawm lub raum cov ntaub so ntswg
PAS staining, Masson staining, thiab hexaammonium silver-hematoxylin-eosin staining tau siv los saib xyuasraum histopathological hloovnyob rau hauv lub teeb microscope thiab kom tau cov duab. Kev xa tawm electron microscopy tau siv los saib xyuas cov kev hloov pauv ntawm lub raum cov ntaub so ntswg thiab cov duab tau sau.

 

2.7 Kev soj ntsuam PCR tiag tiag
Khovcov ntaub so ntswgtau hauv av hauv dej khov thiab TRIzol reagent tau ntxiv rau rho tawm tag nrho RNA, thiab centrifuged ntawm 4 degree nrog lub vojvoog ntawm 9.5 cm. Lub supernatant tau muab rho tawm los ntawm centrifugation ntawm 12 000 r/min rau 10 min, thiab ces centrifuged rau 15 min nrog chloroform. Lub supernatant tau centrifuged dua rau 15 min ntawm chav tsev kub rau 10 min, thiab cov supernatant tau ntxuav nrog 75 feem pua ​​​​ethanol. qhia nyob rau qib mRNA. Cov primers tau tsim los siv Primer Premier 5.0 software thiab synthesized los ntawm Shanghai Bioengineering Co. Ltd. Cov primer sequences yog raws li nram no.