Heyingwuzi formulation alleviates diabetic retinopathy by promoting mitophagy via the HIF-1α/BNIP3/NIX axis

Table 1 Primer sequences used for quantitative reverse transcription-polymerase chain reaction

Gene	Forward primers	Reverse primers
HIF-1α	TGCTCATCAGTTGCCACTTCCAC	CACCCTGTTGCTGTAGCCAAA
BNIP3	AATAATGGGAACGGGGGCAG	CCCCCTTTCTTCATGACGCT
NIX	CACACCAGCAGGGACCATAG	TGTGCTCAGTCGCTTTCCAA
FUNDC1	CCCCCTCCCCAAGACTATGA	TGCAAGTCCGAGCAAAAAGC
PINK1	AGTCCATTGGTAAGGGCTGC	AAATCTGCGATCACCAGCCA
Parkin	AATCAGAGGGCTCTGGAGGT	TCATCCGAATGGACCGGATT
GAPDH	TGACTTCAACAGCGACACCCA	CACCCTGTTGCTGTAGCCAAA

Table 2 Components of the heying wuzi formulation serum that were identified by liquid chromatography-tandem mass spectrometry

No.	Title	RT (min)	Area	Ontology
1	Phosphatidylethanolamine lyso 22	13.94715	1899972.00	2-acyl-sn-glycero-3-phosphoethanolamines
2	Phosphatidylethanolamine lyso 18	15.32940	226900.40	2-acyl-sn-glycero-3-phosphoethanolamines
3	Citric acid	1.820983	184061.70	Tricarboxylic acids and derivatives
4	sn-Glycero-3-phosphocholine	14.45802	144480.10	Glycerophosphocholines
5	(2R,3R,6R,8R,9S,12S,13R,14R,15R,16R)-6,8,14,15-tetrahydroxy-2,6,13,16-tetramethyl-3-{[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-10-oxatetracyclo[7.6.1.0,.0,]hexadec-4-en-11-one	14.30263	143726.20	Eudesmanolides, secoeudesmanolides, and derivatives
6	Phosphatidylethanolamine lyso 20	13.50750	80798.77	2-acyl-sn-glycero-3-phosphoethanolamines
7	2-[(4S,5S,5aS,9aS)-4-methoxy-6,6,9a-trimethyl-5-[(2E,4E,6E)-octa-2,4,6-trienoyl]oxy-1-oxo-4,5,5a,7,8,9-hexahydro-3H-benzo[e]isoindol-2-yl]pentanedioic acid	15.12730	50046.63	Glutamic acid and derivatives
8	Phosphatidylethanolamine lyso alkenyl 16	15.92828	20239.35	Glycerophosphoethanolamines
9	USNIC ACID	7.950984	17973.83	Acetophenones
10	FORMONONETIN	11.89108	16466.15	4’-O-methylisoflavones
11	PFCA-unsaturated	1.420167	15911.72	PFSA
12	3-hydroxy-1a,5-bis(hydroxymethyl)-5,6b-dimethyl-1,3,3a,4,6,6a-hexahydrocyclopropa[e]inden-2-one	11.26097	15753.38	Cyclic alcohols and derivatives
13	4-[5-[[4-[5-[acetyl(hydroxy)amino]pentylamino]-4-oxobutanoyl]-hydroxyamino]pentylamino]-4-oxobutanoic acid	14.49665	426921.60	N-acyl amines
14	LPE 16:0	14.81610	94029.74	Lipids
15	Cytarabine	1.46050	28727.73	Pyrimidine nucleosides
16	Indole-3-acetyl-L-alanine	7.712983	17271.44	Amino acids
17	Taurocholic acid	13.89302	14700.46	Trihydroxy bile acids, alcohols, and derivatives
18	10-Hydroxydecanoic acid	11.39715	11104.43	Medium-chain hydroxy acids and derivatives
19	Phosphatidylethanolamine lyso 16	15.29023	178188.7	2-acyl-sn-glycero-3-phosphoethanolamines
20	Actrarit	6.760533	23273.13	Benzene and substituted derivatives
21	Pyroglutamic acid	1.820983	19421.45	Alpha amino acids and derivatives
22	Atenolol acid	11.30097	12153.77	Phenol ethers
23	D-PANTOTHENIC ACID	1.94130	22119.00	Secondary alcohols
24	Acacetin	18.55157	15547.79	4’-O-methylated flavonoids
25	(1R,6R,10S,11R,13S,15R)-1,6-dihydroxy-8-(hydroxymethyl)-4,12,12,15-tetramethyl-5-oxotetracyclo[8.5.0.0,.0,]pentadeca-3,8-dien-13-yl hexadecanoate	14.02730	61856.96	Phorbol esters
26	arctigenin	11.81143	24300.10	Dibenzylbutyrolactone lignans
27	4-Hydroxy-4-methyl-2-pentanone	20.19918	11647.11	Beta-hydroxy ketones
28	12-hydroxyeicosapentaenoic acid	14.65860	84224.38	Hydroxyeicosapentaenoic acids
29	(+/-)-Synephrine	20.86260	72999.33	1-hydroxy-2-unsubstituted benzenoids
30	Phosphatidylcholine lyso 16	15.93340	59974.72	2-acyl-sn-glycero-3-phosphocholines
31	4-O-Methylphloracetophenone	1.17735	19537.06	Alkyl-phenylketones
32	5-Methoxy-3-indoleacetic acid	8.153967	13901.50	Indole-3-acetic acid derivatives
33	(1R,9S,10S)-3,4-dihydroxy-11,11-dimethyl-5-(propan-2-yl)-16-oxatetracyclo[7.5.2.0,.0,]hexadeca-2(7),3,5-triene-8,15-dione	15.36857	66856.04	Diterpene lactones
34	3-Isobutylglutaric acid	8.214517	16494.50	Methyl-branched fatty acids
35	LPC 18:1	15.80038	1556111.00	Lipids
36	Phosphatidylcholine lyso 18	16.44933	19155.42	2-acyl-sn-glycero-3-phosphocholines
37	Reserpine	15.13208	27432.26	Yohimbine alkaloids
38	ISOPALMITIC ACID	10.76080	133306.00	Long-chain fatty acids
39	Celastrol	16.38930	16379.43	Triterpenoids
40	(4E,8E)-10-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1H-2-benzofuran-5-yl)-4,8-dimethyldeca-4,8-dienoic acid	15.60355	67788.85	Terpene lactones
41	icos-19-ene-1,2,4-triol	11.94487	16965.70	Long-chain fatty alcohols
42	FT-thioether	7.47550	402198.90	PFSA
43	5-Methoxypsoralen	1.379833	717938.30	5-methoxypsoralens
44	Chaulmoogric Acid	13.66750	19960.99	Long-chain fatty acids
45	Cinnamoylglycine	7.752817	16160.29	N-acyl-alpha amino acids
46	Griseofulvin	7.03820	19173.55	Benzofurans
47	Ketoisovaleric acid	1.841267	9078.108	Short-chain keto acids and derivatives
48	Glycocholic Acid	15.75758	439946.10	Glycinated bile acids and derivatives
49	Phosphatidylserine 18	15.56438	30450.24	Phosphatidylserines
50	[(6Z,10Z)-6-(acetyloxymethyl)-10-(hydroperoxymethyl)-3-methylidene-2-oxo-3a,4,5,8,9,11a-hexahydrocyclodeca[b]furan-4-yl] 2-methylbutanoate	9.359233	23463.89	Germacranolides and derivatives
51	(2E,4E)-1-[(2R,6S,14S,22S,25R)-25-(3,3-dimethyloxiran-2-yl)-15-methyl-1,3,13,15-tetraazaheptacyclo[18.4.1.0,.0,.0,.0,.0,]pentacosa-7,9,11,16(21),17,19-hexaen-3-yl]hexa-2,4-dien-1-one	14.41933	93651.37	Alpha carbolines
52	2-[5-[2-[2-[5-(2-hydroxypropyl)oxolan-2-yl]propanoyloxy]propyl]oxolan-2-yl]propanoic acid	12.90287	30013.75	Dicarboxylic acids and derivatives
53	Bufotalin	15.83517	45015.64	Bufanolides and derivatives
54	Furostane base -2H + O-Hex, O-Hex-dHex-dHex-dHex	9.319567	21781.36	Steroidal saponins
55	C18(Plasm)-18:1 PC	11.90607	40133.82	1-(1Z-alkenyl),2-acyl-glycerophosphocholines
56	DErySphingosine	10.83890	38805.35	1,2-aminoalcohols
57	C12-AE1S (TENTATIVE)	12.62238	29299.55	Sulfuric acid monoesters
58	LPC 18:3	13.62767	173150.50	Lipids
59	Nifekalant	6.64155	22225.56	Phenylpropylamines
60	beta-N-Methylaminoalanine	13.10987	30146.63	Alpha amino acids
61	Serine-Cholic Acid	15.32113	551130.30	Glycinated bile acids and derivatives
62	methyl 4-((10R,13R)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl) pentanoate	15.44695	19387.68	Monohydroxy bile acids, alcohols, and derivatives
63	Diatoxanthin	7.272067	14265.04	Triterpenoids
64	Argatroban	15.12730	47349.21	Dipeptides
65	9-Methoxy-2,2-dimethyl-2,6-dihydro-pyrano[3,2-c]quinolin-5-one	1.35255	17009.10	Pyranoquinolines
66	Phosphatidylcholine 16	7.83215	48466.42	Phosphatidylcholines
67	Glutamine	1.33950	40752.10	Alpha amino acids
68	(E)-5-hydroxy-N-[3-[5-[3-[[(E)-5-hydroxy-3-methylpent-2-enoyl]amino]propyl]-3,6-dioxopiperazin-2-yl]propyl]-3-methylpent-2-enamide	11.65172	14764.10	Alpha amino acids and derivatives
69	LPC 16:0	14.89577	1942964.00	Lipids
70	Shanzhiside methyl ester	14.03128	26912.56	Iridoid O-glycosides
71	9-Trans-Palmitelaidic acid	18.88537	58417.61	Long-chain fatty acids
72	Sebacic acid	8.96275	28959.26	Medium-chain fatty acids
73	Vindoline	13.89302	60721.01	Plumeran-type alkaloids
74	9Z,12Z-Linoleic acid (NMR)	19.25802	313966.20	Lineolic acids and derivatives
75	3-Indoxyl sulfate	6.68105	322077.40	Arylsulfates
76	a-Linolenic acid (NMR)	18.11325	80101.46	Lineolic acids and derivatives
77	Arachidonic acid	18.92453	253179.40	Long-chain fatty acids
78	Taurine	8.30815	5742.452	Organosulfonic acids
79	LPC 18:2	14.18563	1163369.00	Lipids
80	Oleic acid	20.54262	181184.10	Long-chain fatty acids
81	Conessine	12.99227	21960.55	Conanine-type alkaloids
82	PROTOVERATRINE A	1.379833	21784.06	Cerveratrum-type alkaloids
83	4-Nitrophenol	20.19397	10567.01	Nitrophenols
84	methyl orsellinate	1.313333	25321.51	P Hydroxybenzoic acid alkyl esters
85	Atalaphylline	13.38752	47479.44	Acridones
86	PFSA-pentafluorosulfide	1.17735	85352.33	PFSA
87	piperazine-2,5-dione	1.841267	12361.39	Alpha amino acids and derivatives
88	N-(4-((6aR,8aS)-4-hydroxy-6a,8a,9-trimethyl-3,4,5,6,6a,6b,7,8,8a,8b,11a,12,12a,12b-tetradecahydro-1H-naphtho[2’,1’:4,5]indeno[2,1-b]furan-10-yl)-2-methylbutyl)acetamide	15.32113	1344867.00	Furostanes and derivatives
89	6-(furan-3-yl)-6,8,12,16,21-pentahydroxy-7,15-dimethyl-9-oxo-3,17,19-trioxaheptacyclo[9.9.3.0,.0,.0,.0,.0,]tricosan-14-yl 2-methylbutanoate	13.50750	38900.18	Limonoids
90	Gedunol	15.79632	305832.30	Limonoids
91	2,6-di-tert-butyl-4-methylphenol	1.70115	164993.80	Phenylpropanes
92	1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine	16.57080	1376141.00	Phosphatidylcholines
93	Trifluoroacetic acid	16.21035	175237.70	PFSA
94	Germinaline	1.379833	26898.38	Alkaloids
95	Phytosphingosine (not validated, isomer of 1696)	10.79983	58379.87	Lipids
96	Choline	15.32113	1686757.00	Cholines
97	Decanoic acid	19.0867	11618.62	Medium-chain fatty acids
98	7-ethenyl-1,4a,7-trimethyl-3,4,6,8,8a,9,10,10a-octahydro-2H-phenanthrene-1-carboxylic acid	17.87777	111714.60	Diterpenoids
99	Uric acid	1.820983	69064.27	Xanthines
100	vanillic acid	1.313333	53861.45	M-methoxybenzoic acids and derivatives
101	Tuberostemonine	12.69258	91277.17	Stichoneurine-type alkaloids
102	(1R,2R,5R,5’S,6S,8aS)-6-(acetyloxy)-2,5,8a-trimethyl-5’’-oxo-octahydro-2H-dispiro[naphthalene-1,2’:5’,3’-bis(oxolane)]-5-ylmethyl acetate	15.32113	668259.40	Diterpene lactones
103	(R)-4-aminoisoxazolidin-3-one	1.934117	30480.44	Alpha amino acids and derivatives
104	(1S,3R,6S,6aR,6bR,8S,9S,11R,11aR,12R,12aR,14R)-1-ethyl-6,8,11-trihydroxy-3-methyl-10-methylenetetradecahydro-3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine 1-oxide	12.69258	247497.70	Kaurane diterpenoids
105	Secoisolariciresinol	12.26407	15677.30	Dibenzylbutanediol lignans
106	Cyclamate	6.110917	33820.46	Cyclamates
107	Docosahexanoic acid	18.55157	591446.80	Very long-chain fatty acids
108	anthothecol	14.03128	23028.57	Limonoids
109	(1R,4aR,5S)-5-(3-hydroxy-3-methylpent-4-enyl)-1,4a-dimethyl-6-methylidene-3,4,5,7,8,8a-hexahydro-2H-naphthalene-1-carboxylic acid	15.60355	517448.60	Diterpenoids
110	(6aR,8aS)-11-(3-acetamido-2-methylpropyl)-6a,8a,9-trimethyl-10-oxo-1,3,4,5,6,6a,6b,7,8,8a,8b,9,10,12,12a,12b-hexadecahydropentaleno[2,1-a]phenanthren-4-yl acetate	14.89468	53006.64	Steroid esters
111	4-((9S)-3,5,14-trihydroxy-10-((E)-((1-hydroxybutan-2-yl)imino)methyl)-13-methylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one	13.89302	42323.20	Cardenolides and derivatives
112	N-ACETYL-L-LEUCINE	7.51500	24397.29	Leucine and derivatives
113	2-Mercaptobenzothiazole	17.58292	35381.59	Benzothiazoles
114	Pristimerin	17.16623	48255.61	Triterpenoids

PFSA: Perfluorosulfonic acid.