Cerebrospinal fluid proteomics reveal potential protein targets of JiaWeiSiNiSan in preventing chronic psychological stress damage

Figures & data

Figure 1. HPLC fingerprint graph of JWSNS. The HPLC fingerprint graph of JWSNS showed 31 common characteristic peaks: one peak (1) belonged to Rehmannia glutinosa; one peak (7) belonged to Lycium barbarum L.; eight peaks (3, 4, 5, 6, 9, 10, 20, 23 and 25) belonged to Gardenia jasminoides Ellis; eight peaks (2, 8, 12, 13, 14, 16, 20 and 27) belonged to Paeonia lactiflora; 14 peaks (11, 15, 17, 18, 19, 20, 21, 22, 24, 26, 28, 29, 30 and 31) belonged to Aurantii Fructus. Compared with reference substance, peaks 10, 14, 18 and 19 were respectively identified as geniposide, paeoniflorin, naringin, hesperidin.

Table 1. The table of components in JiaWeiSiNiSan.

Plant species	Family	Plant part	Pinyin
Bupleurum chinense DC.	Apiaceae	Root	Chai hu
Paeonia lactiflora Pall.	Paeoniaceae	Root	Bai shao
Citrus × aurantium L.	Rutaceae	Fruit	Zhi qiao
Lycium barbarum L.	Solanaceae	Fruit	Gou qi
Gardenia jasminoides J.Ellis	Rubiaceae	Fruit	Zhi zi
Rehmannia glutinosa Seud.	Scrophulariacea	Rootstock	Di huang
Haliotis diversicolor Reeve	Haliotidae	Shell	Shi jue ming

Figure 2. Experiment procedure.

Figure 3. Behavioural test results. (A) Result of Sucrose Preference Test (SPT); (B) result of Open Field Test (OFT); (C) result of Forced Swimming Test (FST); (D) result of T-Maze. Data were expressed as the mean ± SEM, *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01.

Figure 4. Amount of resilient, susceptible, non-sensitive rats of CUMS and JESNS group in Sucrose Preference Test (SPT) (A), Open Field Test (OFT) (B) and Forced Swimming Test (FST) (C); and (D) amount of rats of CUMS and JWSNS group that showed resilience in one, two and all above behavioural tests.

Figure 5. GO annotation and KEGG pathway enrichment analysis of Differently expressed proteins (DEPs). (A) GO annotation of DEPs in CON and CUMS group; (B) KEGG pathway enrichment analysis of DEPs in CON and CUMS group; (C) GO annotation of DEPs in CUMS and JWSNS group; (D) KEGG pathway enrichment analysis of DEPs in CUMS and JWSNS group.

Figure 6. (A) Venn map of differentially expressed proteins co-regulated by JWSNS and CUMS; (B) Protein–Protein Interaction Network (PPI) of co-regulated DEPs.

Table 2. Differentially expressed proteins co-regulated by JWSNS and CUMS.

Protein IDs	protein_name	gene_id	gene_name	CUMS vs JWSNS	CUMS vs CON
ENSRNOP00000069539	MIA SH3 domain ER export factor 3-	ENSRNOG00000059202	AABR07021988.1	1.667021	1.406647
ENSRNOP00000014807	Insulin-like growth factor binding protein 6	ENSRNOG00000010977	Igfbp6	1.240446	1.284354
ENSRNOP00000012293	Alpha-2u globulin PGCL3	ENSRNOG00000009273	LOC259244	1.289872	1.277781
ENSRNOP00000027860	HtrA serine peptidase 1	ENSRNOG00000020533	Htra1	1.227982	1.246935
ENSRNOP00000004764	EGF containing fibulin extracellular matrix protein 1	ENSRNOG00000003553	Efemp1	1.239637	1.212169
ENSRNOP00000033950	Ubiquitin-like modifier activating enzyme 1	ENSRNOG00000019164	Uba1	0.607629	0.787971
ENSRNOP00000017234	Heparin binding growth factor	ENSRNOG00000042261	Hdgf	0.526065	0.771831
ENSRNOP00000004867	Small ubiquitin-like modifier 2	ENSRNOG00000003670	Sumo2	0.558033	0.746357
ENSRNOP00000074688	Ubiquitin C	ENSRNOG00000057823	Ubc	0.667817	0.733036
ENSRNOP00000015598	RAB11a, member RAS oncogene family	ENSRNOG00000011302	Rab11a	0.70795	0.730942
ENSRNOP00000009556	heat shock protein HSP 90-alpha	ENSRNOG00000007219	LOC103692716	0.675043	0.730779
ENSRNOP00000024430	Vimentin	ENSRNOG00000018087	Vim	0.797659	0.727629
ENSRNOP00000009649	Proteasome 26S subunit, ATPase 6	ENSRNOG00000007203	Psmc6	0.620382	0.718329
ENSRNOP00000001518	Ribosomal protein lateral stalk subunit P0	ENSRNOG00000001148	Rplp0	0.656478	0.696513
ENSRNOP00000072016	TATA-box binding protein associated factor 15	ENSRNOG00000058393	Taf15	0.667331	0.693424
ENSRNOP00000070868	Tubulin, alpha 1B	ENSRNOG00000053468	Tuba1b	0.588112	0.689164
ENSRNOP00000026696	Heat shock protein family A member 9	ENSRNOG00000019525	Hspa9	0.535606	0.679796
ENSRNOP00000019247	Ribosomal protein L27a	ENSRNOG00000014214	Rpl27a	0.692805	0.673251
ENSRNOP00000071233	Spectrin, beta, non-erythrocytic 1	ENSRNOG00000005434	Sptbn1	0.736569	0.670589
ENSRNOP00000022603	Calmodulin 3	ENSRNOG00000016770	Calm3	0.526308	0.636832
ENSRNOP00000010674	Tyrosyl-tRNA synthetase	ENSRNOG00000007213	Yars	0.54097	0.556245
ENSRNOP00000009249	Proteasome 26S subunit, non-ATPase 6	ENSRNOG00000006751	Psmd6	0.46405	0.550629
ENSRNOP00000056260	40S ribosomal protein S14-like	ENSRNOG00000018774	LOC100911847	0.534893	0.523183
ENSRNOP00000038448	Seryl-tRNA synthetase	ENSRNOG00000020255	Sars	0.479899	0.517425
ENSRNOP00000071398	Glutaminyl-tRNA synthetase	ENSRNOG00000060912	Qars	0.587713	0.514999
ENSRNOP00000070867	Neuraminidase 1	ENSRNOG00000032942	Neu1	0.550484	0.48937
ENSRNOP00000001397	Transmembrane p24 trafficking protein 2	ENSRNOG00000001053	Tmed2	0.446784	0.473675
ENSRNOP00000070331	SET nuclear proto-oncogene	ENSRNOG00000025892	Set	0.431938	0.455886
ENSRNOP00000026528	Ribosomal protein S5	ENSRNOG00000019453	Rps5	0.354136	0.414189
ENSRNOP00000034657	Similar to Finkel-Biskis-Reilly murine sarcoma virusubiquitously expressed	ENSRNOG00000047365	LOC687780	0.401339	0.392441
ENSRNOP00000033144	Ribosomal protein s25	ENSRNOG00000027503	Rps25	0.320287	0.364721
ENSRNOP00000004278	Ribosomal protein S4, X-linked	ENSRNOG00000003201	Rps4x	0.3718	0.353676
ENSRNOP00000022184	Ribosomal protein S12	ENSRNOG00000016411	Rps12	0.227142	0.255554
ENSRNOP00000060949	Ribosomal protein L34	ENSRNOG00000016387	Rpl34	0.145147	0.150869
ENSRNOP00000074005	Dyskerin pseudouridine synthase 1	ENSRNOG00000055562	Dkc1	0.153169	0.13148

Table 3. PRM quantitative analysis of target proteins.

Protein name	Gene name	PRM results		TMT results
		CUMS/CON	JWSNS/CUMS	CUMS/CON	JWSNS/CUMS
ribosomal protein S4, X-linked	Rps4x	3.4306	0.6859	2.8274	0.3718
heat shock protein HSP 90-alpha	HSP90AA1	3.6841	0.2989	1.3684	0.6750
ribosomal protein S12	Rps12	6.4356	0.3992	3.9131	0.2271
ubiquitin-like modifier activating enzyme 1	Uba1	2.3242	0.5349	1.2691	0.6076
ribosomal protein S14	Rps14	15.7618	0.1425	1.9114	0.5349
tubulin, alpha 1B	Tuba1b	2.8018	0.6105	1.4510	0.5881

Figure 7. (A) BrdU/DCX positive cells in DG, red: BrdU, green: DCX, blue: DAPI. (B) Number of BrdU/DCX positive cells in DG (n = 5 per group). Data were expressed as the mean ± SEM. *p < 0.05, **p < 0.01, vs. control. #p < 0.05, ##p < 0.01, vs. CUMS.

Figure 8. (A) NeuN positive cells in DG, green: NeuN, blue: DAPI. (B) Number of neurons in DG (n = 5 per group). Data were expressed as the mean ± SEM. *p < 0.05, **p < 0.01, vs. control. #p < 0.05, ##p < 0.01, vs. CUMS.

Data availability statement

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD021735. The mass spectrometry proteomics data have been also deposited to the iProX with the dataset identifier PXD021776.