Ntu Peb DsbA-L cuam tshuam nrog VDAC1 hauv Mitochondrionmediated Tubular Cell Apoptosis Thiab Pab Txhawb rau Kev Loj Hlob Ntawm Kab Mob Raum Mob

7. tiam PT-VDAC1-KO nas

Txhawm rau tshawb xyuas lub luag haujlwm ntawm VDAC1 hauv AKI, peb tau tsim tsa tus qauv nas uas muaj qhov sib tw ntawm VDAC1 hauv cov tubules ze ntawm lub raum. Daim duab 7a qhia txog kev cog qoob loo siv los tsim PT-VDAC1-KO. RT-PCR tau siv los genotype PT- VDAC1-KO nas; qhov no tau ua tiav los ntawm kev nthuav dav ib qho 416-bp DNA fragment floxed allele thiab ib 370-bp DNA fragment ntawm Cre gene (Daim duab 7, b; txoj kab 4 thiab 6). Cov nas qus (PT-VDAC1-WT) tsis muaj Cre gene (Daim duab 7, b, kab 2 thiab 3). Kev tshuaj ntsuam Immunoblot tau qhia tias qib kev qhia ntawm VDAC1 hauv lub raum cortices ntawm PTVDAC1-KO nas tau qis dua cov nyob hauv PTVDAC1-WT nas tom qab kev kho mob ischemic lossis sham (Daim duab 7 c thiab d). Qhov no tau raug txheeb xyuas ntxiv los ntawm kev siv tshuaj tiv thaiv kab mob ntawm VDAC1 (Daim duab 7e). Cov ntaub ntawv no tau txheeb xyuas tias peb tau ua tiav ib qho PT- VDAC1-KO nas qauv.

Daim duab 7. Tsim thiab yam ntxwv ntawm PT-VDAC1-KO nas qauv. (a, b) Cov txheej txheem yug me nyuam rau tiam ntawm PTVDAC1-KO nas thiab PCR nrhiav pom ntawm genotype ntawm cov tsiaj qus thiab floxed alleles ntawm VDAC1 thiab PEPCK-Cre allele. (c) Kev tshuaj ntsuam immunoblot ntawm VDAC1 thiab b-tubulin siv raum cortical cov ntaub so ntswg ntawm PT-VDAC1-KO thiab PT-VDAC1-WT littermate nas tom qab IR raug mob. (d) Kev tshuaj xyuas ntawm cov duab grayscale nruab nrab ntawm lawv. (e) Immunohistochemical staining ntawm VDAC1 hauv lub raum cortical cov ntaub so ntswg ntawm cov tsiaj qus thiab VDAC1 nas tom qab IR raug mob. Thawj magnification x 400. Scar Bar: 100µM. Txhua qhov kev sim (c & e) tau rov ua dua 6 zaug ntawm nws tus kheej nrog cov txiaj ntsig zoo sib xws. Ib txoj kev ANOVA tau siv rau ntau pawg sib piv (d). #p<0.05: versus saline group, sham group, or MCD group. # P<0.05 versus sham group. * P<0.05 versus PT-VDAC1-WT with IR group.

Nyem qhov no kom paub cov txiaj ntsig Cistanche

8. I/R-induced raum raug mob, tubular cell apoptosis, thiab mitochondrial puas tau ameliorated hauv PTVDAC1-KO nas

Littermates ntawm PT-VDAC1-WT thiab PT- VDAC1-KO nas tau kho nrog ischemia (28 min) tom qab ntawd los ntawm kev raug mob rov qab (48 h). Tom qab ntawd peb tau txais cov qauv ntshav thiab lub raum cov ntaub so ntswg rau kev kuaj ntxiv. Peb tau pom tias PT-VDAC1-KO tseem ceeb cuam tshuam qhov kev nce siab ntawm I/R-induced hauv qib BUN thiab creatinine (Daim duab 8 a thiab b). Thib ob, histological thiab TUNEL kev soj ntsuam ntxiv tau pom tias PT-VDAC1-KO tau ua rau muaj kev puas tsuaj rau cov ntaub so ntswg I/R thiab lub raum apoptosis (Daim duab 8 c, d, f, thiab g). Thib peb, EM tsom xam pom tias PT-VDAC{15}}KO tau txo qis I/R-induced mitochondrial puas tsuaj (Daim duab 8 e thiab h). Thaum kawg, immunoblotting tau lees paub tias PT-VDAC1-KO nas pom tau tias txo qis ntawm cleaved caspase-3, qis dua ntawm Bax hauv mitochondria, thiab qis dua ntawm Cyt-c tso rau hauv cytosol (Daim duab 8 ib-o). Cov ntaub ntawv no tau qhia tias VDAC1 kuj tau kho qhov I / R-induced AKI.

Daim duab 8. Attenuation ntawm IR-induced raum raug mob, tubular cell apoptosis, mitochondrion puas, thiab kev qhia ntawm Bax, Cyt-c, thiab cleaved caspase3 hauv PT-VDAC1-KO nas. Ob lub raum cov hlab ntsha ntawm PT-VDAC1-KO thiab PT-VDAC1-WT cov nas littermate tau clamped rau 28 feeb thiab tom qab ntawd tso tawm rau 48 teev los tsim kom muaj tus qauv IR. (a) IB. (b) Serum creatinine. (c) Hematoxylin thiab eosin staining. (d) Cov chaw sawv cev ntawm TUNEL-zoo hlwb. (e) Electron microscope nrhiav pom ntawm mitochondrion morphology. (f) Tubular puas tau qhab nia. (g) Tus naj npawb ntawm TUNEL-zoo hlwb. (h) Mitochondria raug mob qhab nia. (i) Cov immunoblots txheeb xyuas qhov kev qhia ntawm cleaved caspase3, VDAC1, thiab DsbA-L nrog rau kev qhia ntawm BAX thiab Cyt-c hauv mitochondrial thiab cytosolic feem. b-tubulin thiab GAPDH tau siv tag nrho lysate lossis cytosolic loading tswj, feem. COX IV tau siv los ua mitochondria loading tswj. (j-p) Kev tsom xam ntawm cov duab grayscale nruab nrab ntawm lawv. Thawj magnification x 400. Scar Bar: 100µM hauv c & d 20µM hauv e. Txhua qhov kev sim (c- e&i) tau rov ua dua 6 zaug ntawm nws tus kheej nrog cov txiaj ntsig zoo sib xws. Ib txoj kev ANOVA tau siv rau ntau pawg sib piv (a, b, f- h&j- p). #p<0.05: versus saline group, sham group, or MCD group. # P<0.05 versus sham group. * P<0.05 versus PT-VDAC1 WT with IR group.

Cistanche tubulosa

9. Qhov overexpression ntawm DsbA-L aggravated I/R-induced raum puas tau attenuated nyob rau hauv PT-VDAC1-KO nas

DsbA-L thaum raug mob ischemic. Ib lub lis piam ua ntej tus qauv nas ntawm IR tau tsim, peb tau txhaj cov leeg ntawm txhua tus nas nrog DsbA-L plasmid (kev txhaj tshuaj tau ua ob zaug hauv lub lim tiam no). Qhov overexpression ntawm DsbA-L aggravated IR-induced elevation nyob rau hauv BUN thiab creatinine qhia, raum puas, thiab raum apoptosis; Cov teebmeem no tau txo qis hauv PTVDAC1-KO nas (Daim duab 9 a-d, e, thiab f). Cov txiaj ntsig ntawm Immunoblot tau qhia ntxiv tias qhov kev tshaj tawm ntawm DsbA-L tau txhim kho IR-induced nce hauv cleaved caspase -3, tsub zuj zuj ntawm Bax hauv mitochondria, thiab tso tawm Cyt-c rau hauv cytosol; Cov teebmeem no tau txo qis hauv PT-VDAC1-KO nas (Daim duab 9 g thiab h- l). Sib sau ua ke, cov txiaj ntsig no tau pom ntxiv tias VDAC1 kho cov nyhuv ntawm DsbA-L thaum raug mob ischemic.

Daim duab 9. Qhov overexpression ntawm DsbA-L aggravated I/R-induced raum puas tau attenuated nyob rau hauv PT-VDAC1-KO nas. PT-VDAC1-KO thiab PT-DsbA-L-WT nas tau kho nrog lossis tsis muaj DsbA-L plasmid thiab tom qab ntawd tsim qauv IR. (a) PAB. (b) Serum creatinine. (c) Hematoxylin thiab eosin staining. (d) Cov chaw sawv cev ntawm TUNEL-zoo hlwb. (e) Tubular puas qhab nia. (f) Tus naj npawb ntawm TUNEL-zoo hlwb. (g) Cov tshuaj tiv thaiv kab mob tau tshuaj xyuas qhov kev qhia ntawm cleaved caspase3, VDAC1, thiab DsbA-L nrog rau kev qhia ntawm BAX thiab Cyt-c hauv mitochondrial thiab cytosolic feem. b-tubulin thiab GAPDH tau siv tag nrho lysate lossis cytosolic loading tswj, feem. COX IV tau siv los ua mitochondria loading tswj. (h-n) Kev tsom xam ntawm cov duab grayscale nruab nrab ntawm lawv. Thawj magnification x 400. Scar Bar: 100mM. Txhua qhov kev sim (c-e) tau rov ua dua 6 zaug ntawm nws tus kheej nrog cov txiaj ntsig zoo sib xws. Ib txoj kev ANOVA tau siv rau ntau pawg sib piv (a, b, thiab f- n). #p<0.05: versus saline group, sham group, or MCD group. # P<0.05 versus sham group. ^ P<0.05 versus IR group.* P<0.05 versus IR group.

Cistanche ntxiv

10. Lub attenuation ntawm I/R-induced raum puas hauv PTDsbA-L-KO nas raug txo los ntawm overexpression ntawm VDAC1

Txhawm rau kom paub meej ntxiv yog tias DsbA-L-mediated tubular kev puas tsuaj yog nyob ntawm VDAC1, cov nas littermate ntawm PTDsbA-L -WT thiab PT- DsbA-L -KO nas tau pom thawj zaug rau kev kho I / R, thiab tom qab ntawd txhaj tshuaj ntawm tus Tsov tus tw leeg. nrog VDAC1 overexpression plasmid. Qhov I / R-induced nce nyob rau hauv BUN thiab creatinine theem tau ameliorated hauv PT- DsbA-L -KO nas; Txawm li cas los xij, qhov kev cuam tshuam no tau tiv thaiv los ntawm overexpression ntawm VDAC1 (Daim duab 10 a thiab b). Nws thiab TUNEL staining pom tias cov txheeb ze ntawm IR-induced lub raum cov ntaub so ntswg puas thiab lub raum cell apoptosis raug txo nyob rau hauv PT- DsbAL -KO nas thiab hais tias cov teebmeem no tau thaiv los ntawm overexpression ntawm VDAC1 (Daim duab 10 c-f). Immunoblotting tau pom tias PT- DsbA-L -KO nas muaj qis dua ntawm cleaved caspase-3, qis qis ntawm Bax tsub zuj zuj hauv mitochondria, thiab qis qis ntawm Cyt-c tso rau hauv cytosol; Txawm li cas los xij, cov teebmeem no tau raug tshem tawm los ntawm kev tshaj tawm ntawm VDAC1 (Daim duab 10 g-m). Cov ntaub ntawv no tau txhawb nqa qhov tseeb tias DsbA-L txhawb kev raug mob ischemic nyob rau hauv ib yam uas nyob ntawm VDAC1.

Daim duab 10. PT-DsbA-L-KO ameliorated IR-induced nas raum raug mob tau ploj zuj zus los ntawm overexpression ntawm VDAC1. Cov PT-DsbA-L-KO thiab PT- DsbA-L-WT nas tau kho nrog lossis tsis muaj VDAC1 plasmid thiab tom qab ntawd tsim qauv IR. (a) IB. (b) Serum creatinine. (c) Hematoxylin thiab eosin staining. (d) Cov chaw sawv cev ntawm TUNEL-zoo hlwb. (e) Tubular puas qhab nia. (f) Tus naj npawb ntawm TUNEL-zoo hlwb. (g) Cov tshuaj tiv thaiv kab mob tau tshuaj xyuas qhov kev qhia ntawm cleaved caspase3, VDAC1, thiab DsbA-L nrog rau kev qhia ntawm BAX thiab Cyt-c hauv mitochondrial thiab cytosolic feem. b-tubulin thiab GAPDH tau siv tag nrho lysate lossis cytosolic loading tswj, feem. COX IV tau siv los ua mitochondria loading tswj. (hm) Kev tsom xam ntawm cov duab grayscale nruab nrab ntawm lawv. Thawj magnification x 400. Scar Bar: 100mM. Txhua qhov kev sim (c, d, thiab g) tau rov ua dua 6 zaug ntawm nws tus kheej nrog cov txiaj ntsig zoo sib xws. Ib txoj kev ANOVA tau siv rau ntau pawg sib piv (a, b, e, f, thiab h-m). #p<0.05: versus saline group, sham group, or MCD group. # P<0.05 versus sham group. * P<0.05 versus IR group.

Herba Cistanche

11. CLP-induced lub raum raug mob tau attenuated hauv PT-DsbA-LKO lossis PT-VDAC1-KO nas

Sepsis-induced AKI yog lwm qhov ua rau AKI hauv kev kho mob. Littermates ntawm PT- DsbA-L lossis VDAC-WT thiab PT-DsbA-L lossis VDAC1-KO tau raug rau CLP qauv thiab tom qab ntawd euthanized 18 h tom qab kev phais. Cov txiaj ntsig tau qhia tias PT-DsbA-L lossis VDAC1- KO nas tau txo qis CLP-induced elevations hauv BUN thiab creatinine qib, thiab nce qib ntawm lub raum puas tsuaj thiab lub raum cell apoptosis (Figures S1 thiab S2 a-f ). Immunoblotting tau lees paub tias PTDsbA-L lossis VDAC1-KO nas kuj pom tau tias txo qis ntawm cleaved caspase-3, qis qis ntawm Bax tsub zuj zuj hauv mitochondria, thiab qis dua ntawm Cyt-c tso rau hauv cytosol (Figures S1 thiab S2 g-n). Sib sau ua ke, cov ntaub ntawv no tau pom tias ob qho tib si DsbA-L thiab VDAC1 kho kom haum rau kev nce qib ntawm septic AKI.

12. VAN-induced lub raum raug mob tau attenuated nyob rau hauv PT-DsbAL-KO los yog PT-VDAC1-KO nas

Vancomycin (VAN)-induced AKI kuj yog tus qauv tseem ceeb rau AKI. Cov littermates ntawm PT- DsbA-L los yog VDAC1-WT thiab PT-DsbA-L los yog VDAC1-KO tau txhaj tshuaj intraperitoneally nrog VAN (600mg / kg) rau 7 hnub sib law liag. Cov txiaj ntsig tau qhia tias PT-DsbA-L lossis VDAC1-KO nas tau txo qis VAN-induced nce qib ntawm BUN thiab creatinine, raum cov ntaub so ntswg puas, thiab raum cell apoptosis (Figures S3 thiab S4 an- f). Immunoblot soj ntsuam ntxiv tau lees paub tias PTDsbA-L lossis VDAC1-KO nas kuj tau pom qhov txo qis ntawm cleaved caspase-3, qis dua ntawm Bax tsub zuj zuj hauv mitochondria, thiab qis dua ntawm Cyt-c tso rau hauv cytosol (Figures S3 thiab S4 g-m). Hauv cov ntsiab lus, cov ntaub ntawv no tau pom tias ob qho tib si DsbA-L thiab VDAC1 mediated VAN-induced AKI.

Cistanche extract

13. DsbA-L mediated I/R induced HK-2 cells apoptosis tau txuam nrog VDAC1

Cov txiaj ntsig saum toj no pom tias DsbA-L tau koom nrog hauv BUMPT hlwb apoptosis, txawm li cas los xij, kev ua haujlwm ntawm DsbA hauv tib neeg lub raum tubular hlwb tseem tsis meej. DsbA-L siRNA tau kis mus rau HK2 hlwb thiab tom qab ntawd raug rau ATP depletion rau 2 h thiab rov qab rau 2 h. Cov txiaj ntsig ntawm immunoblot tau pom tias DsbA-L siRNA tshwj xeeb yog ameliorated I / R-induced nce hauv cleaved caspase-3 thiab VDAC1 qhia, qhov nce ntawm Bax nyob rau hauv mitochondria, thiab nce ntau lawm ntawm Cyt-c rau hauv cytosol ( Daim duab S5 an-h). Cov ntaub ntawv kuj tau qhia tias lub luag haujlwm ntawm DsbA-L hauv HK2 cell apoptosis muaj feem xyuam nrog VDAC1 thaum kho I / R.

14. DsbA-L mediated I/R induced HK-2 cells apoptosis yog nyob ntawm VDAC1

Txhawm rau txheeb xyuas ntxiv seb lub luag haujlwm ntawm DsbA-L hauv HK2 cell apoptosis puas yog nyob ntawm VDAC1. Ua ntej, HK2 hlwb tau sib txuas nrog DsbA-L siRNA ntxiv nrog lossis tsis muaj VDAC1 plasmid thiab tom qab ntawd raug rau ATP depletion rau 2 h thiab rov qab rau 2 h. Cov txiaj ntsig ntawm cov tshuaj tiv thaiv kab mob tau qhia tias DsbA-L siRNA ua rau muaj kev cuam tshuam ntau ntxiv ntawm I / R-induced hauv cleaved caspase-3 thiab VDAC1 kev qhia, nce ntxiv ntawm Bax hauv mitochondria, thiab nce ntau ntawm Cyt-c rau hauv cytosol, Cov nyhuv no tau thim rov qab los ntawm kev tshaj tawm ntawm VDAC1 plasmid (Daim duab S6a-h). Qhov thib ob, HK2 hlwb tau koom ua ke nrog VDAC1 siRNA ntxiv nrog lossis tsis muaj DsbA-L plasmid thiab tom qab ntawd raug rau kuv (2 h) / R (2 h). Cov txiaj ntsig ntawm cov tshuaj tiv thaiv kab mob tau pom tias VDAC1 siRNA tshwj xeeb yog ameliorated I / R-induced nce hauv cleaved caspase-3 thiab VDAC1 qhia, nce ntxiv ntawm Bax nyob rau hauv mitochondria, thiab nce ntau lawm ntawm Cyt-c rau hauv cytosol, cov nyhuv no. tsis tau txhim kho los ntawm kev tshaj tawm ntawm DsbA-L plasmid (Daim duab S7a-h). Cov ntaub ntawv tau lees paub meej tias VDAC1 tau kho lub luag haujlwm pro-apoptosis ntawm DsbA-L hauv HK-2 hlwb thaum kho I/R.

Xiaozhou Li,a,b,1 Jian Pan,a,b,1 Huiling Li,c Guangdi Li,g Bohao Liu,a,b Xianming Tang,a,b Xiangfeng Liu,f Zhibiao He,a,b Zhenyu Peng,a , b Hongliang Zhang, a, b Luxiang Wang, a, b Yijian Li, d Xudong Xiang, a, b Xiangping Chai, a, b Yunchang Yuan, e Peilin Zheng, h thiab Dongshan Zhang a, b *

ib lub Department of Emergency Medicine, People's Republic of China

b Emergency Medicine and Difficult Diseases Institute, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, People's Republic of China

c Department of Ophthalmology, Tib neeg koom pheej ntawm Tuam Tshoj

d Department of Urinary Surgery, People's Republic of China

e Department of Chest Surgery, People's Republic of China

f Department of General Surgery, Second Xiangya Hospital, People's Republic of China

g Department of Public Health, Central South University, Changsha, Hunan, Tib neeg koom pheej ntawm Tuam Tshoj

h Department of Endocrinology, Shenzhen Tib Neeg Lub Tsev Kho Mob, Lub Tsev Kawm Ntawv Kho Mob Thib Ob ntawm Jinan University, Thawj Lub Tsev Kho Mob ntawm Southern University of Science thiab Technology, Shenzhen, Tib Neeg Lub Tebchaws ntawm Tuam Tshoj

1 Xiaozhou Li thiab Jian Pan tau pab tib yam rau txoj kev kawm no