Validation of Jianpi Qingre Tongluo Recipe in Reducing Inflammation and Dyslipidemia in Osteoarthritis via Lnc RNA HOTAIR/APN/PI3K/AKT

Figures & data

Table 1 Clinical Characteristics of Subjects

Index	HC (n=20)	OA (n=30)
		Before Treatment	After Treatment
Gender (female)	12	17^c	17^c
Age (years)	54.65 ± 9.35	55.87 ± 10.06^d	55.87 ± 10.06^a
ESR (mm/h)	9.35 ± 5.86	17.5 (8.0, 32.5)^b**	20 (10.75, 30.25)^b
hs-CRP (mg/L)	1.34 (0.89, 3.96)	1.62 (0.8, 7.73)^b	1.53 (0.36, 4.31)^b#
TC (mmol/L)	2.01 ± 0.67	4.75 (3.91, 5.45)^b**	4.66 (4.02, 5.25)^b
TG (mmol/L)	0.62 ± 0.41	1.4 (1.15, 1.68)^b**	1.07 (0.74, 1.22)^b##
IgA (g/L)	1.77 ± 0.64	2.33 ± 0.85^d*	2.30 ± 0.89^a
IgG (g/L)	8.21 (7.96, 9.30)	9.58 (7.68, 10.72)^b	8.99 (7.8, 10.78)^b
IgM (g/L)	0.89 (0.54, 1.78)	1.14 (0.76, 1.56)^b	1.02 (0.75, 1.51)^b
C3 (g/L)	1.07 (0.96, 1.10)	1.13 ± 0.18^b*	1.14 ± 0.19^a
SDS score	NA	51.79 ± 8.42	50.6 ± 6.25^a
VAS score	NA	6 (5, 6)	1 (1, 2)^b##
SAS score	NA	42.5 (35.93, 47.5)	40 (37.18, 43.75)^b#

Notes: Clinical characteristics of subjects. ^aStudent’s t-test. ^bNonparametric test (median). ^cChi-square test. ^dTwo independent samples t-test. *P < 0.05, **P < 0.01 vs HC group; ^#P < 0.05, ^##P < 0.01, vs before treatment.

Abbreviations: HC, healthy control; OA, Osteoarthritis; hs-CRP, hyper-sensitivity C-reactive protein; C3, complement C3; IgA, G, M, immunoglobulin A, G, M; ESR, erythrocyte sedimentation rate; TC, Total cholesterol; TG, Triglyceride; SAS, Self Rating Anxiety Scale; SDS, Self Rating Depression Scale; VAS, visual analogue scale. NA, not applicable.

Figure 1 Expression of HOTAIR and changes in inflammation and APN. (A) RT-qPCR to test HOTAIR expression. (B–D) ELISA to analyze TNF-α, IL-10, and APN levels. All experiments were replicated thrice. *P < 0.05, **P < 0.01.

Abbreviations: NC, Control group; HOTAIR, Lnc RNA HOTAIR; APN, Adiponectin; TNF-α, Tumornecrosisfactor-α; IL-10, Interleukin-10.

Table 2 Association Rule Analysis of JQP with Clinical Characteristics of OA Patients

The Former	The Latter	Support (%)	Confidence (%)	Lift
JQP	hs-CRP ↓	20.00	71.42	1.42
JQP	C3↓	24.00	85.71	1.33
JQP	TNF-α↓	18.00	70.42	1.01
JQP	IgA↓	20.00	71.42	1.27
JQP	IgM↓	20.00	71.42	1.08
JQP	IgG↓	18.00	64.28	1.07
JQP	C4↓	18.00	64.28	1.10
JQP	TC↓	16.00	57.14	1.02

Note: Association rule analysis of JQP with clinical characteristics of OA patients.

Abbreviations: JQP, Jianpi Qingre Tongluo Prescription; hs-CRP, hyper-sensitivity C-reactive protein; C3, complement C3; C4, complement C4; IgA, G, M, immunoglobulin A, G, M; TC, Total cholesterol; TNF-α, Tumornecrosisfactor-α.

Table 3 The Random Walk Evaluation Model of Laboratory Indicators of OA Patients Treated with JQP

Indicators	Maximum Random Volatility	Walking Steps	Positive Walk Growth Rate	Positive Walk Growth Rate	Improvement Factor	Ratio
IgA	35	287	0.122	0.2066±0.0759	0.4167	8.2
IgM	16	12	1.3333	0.3525±0.1946	0.1905	0.75
IgG	27	287	0.0941	0.2843±0.1373	0.3214	10.63
C4	23	287	0.0801	0.1874±0.1459	0.2738	12.48

Note: The random walk evaluation model of laboratory indicators of OA patients treated with JQP.

Abbreviations: C4, complement C4; IgA, G, M, immunoglobulin A, G, M.

Figure 2 Random walking analysis of JQP and clinical indicators.

Abbreviations: JQP, Jianpi Qingre Tongluo Prescription; C4, complement C4; IgA, G, M, immunoglobulin A, G, M.

Table 4 Active Ingredient Information of JQP

MOL ID	Molecular	OB (%)	DL
MOL001371	Populoside_qt	108.89	0.2
MOL002934	NEOBAICALEIN	104.34	0.44
MOL001351	Gibberellin A44	101.61	0.54
MOL001348	Gibberellin 17	94.64	0.49
MOL001353	GA60	93.17	0.53
MOL002937	DIHYDROOROXYLIN	66.06	0.23
MOL004561	Sudan III	84.07	0.59
MOL001360	GA77	87.89	0.53
MOL001340	GA120	84.85	0.45
MOL001339	GA119	76.36	0.49
MOL002932	Panicolin	76.26	0.29
MOL012246	5,7,4’-trihydroxy-8-methoxyflavanone	74.24	0.26
MOL001342	GA121-isolactone	72.7	0.54
MOL002927	Skullcapflavone II	69.51	0.44
MOL001358	Gibberellin 7	73.8	0.5
MOL002911	2,6,2’,4’-tetrahydroxy-6’-methoxychaleone	69.04	0.22
MOL001349	4a-formyl-7alpha-hydroxy-1-methyl-8-methylidene-4aalpha,4bbeta-gibbane-1alpha,10beta-dicarboxylic acid	88.6	0.46
MOL001344	GA122-isolactone	88.11	0.54
MOL001329	2,3-didehydro GA77	88.08	0.53
MOL001361	GA87	68.85	0.57

Figure 3 Venn diagram of the target genes for JQP and OA.

Figure 4 Network of Drug-Compound-Target of JQP. LGT: Tripterygium wilfordii Hook. f, WLX: Clematis chinensis Osbeck, ZZ: Gardenia jasminoides J. Ellis, HQIN: Scutellaria baicalensis Georgi, WG: Scolopendra subspinipes mutilans L. Koch, YYR: Coix lacryma-jobi L, TR: Juglans regia L, HQ: Astragalus mongholicus Bunge. Green arrow nodes: the eight Chinese herbal medicines in JQP; Orange diamond nodes: potential drug targets; pink octagon nodes: active compounds.

Table 5 The Core Active Ingredient of JQP for the Treatment of OA

Chinese Medicine Name	Mol ID	Molecule Name	Degree
Gardenia jasminoides J.Ellis, Astragalus mongholicus Bunge	MOL000098	Quercetin	254
Gardenia jasminoides J.Ellis, Astragalus mongholicus Bunge, Tripterygium wilfordii Hook. f	MOL000422	Kaempferol	150
Scutellaria baicalensis Georgi, Clematis chinensis Osbeck, Gardenia jasminoides J.Ellis, Juglans regia L, Tripterygium wilfordii Hook. f	MOL000358	Beta-sitosterol	135
Scutellaria baicalensis Georgi, Clematis chinensis Osbeck, Gardenia jasminoides J.Ellis, Coix lacryma-jobi L, Tripterygium wilfordii Hook. f	MOL000449	Stigmasterol	115
Juglans regia L, Astragalus mongholicus Bunge	MOL000296	Hederagenin	45

Table 6 The Core Targets of JQP in OA Treatment

Gene Name	Target Name	Degree
TNF	Tumor necrosis factor	88
IL6	Interleukin-6	87
IL1B	Interleukin-1 beta	82
AKT1	Serine/threonine-protein kinase AKT	81

Figure 5 Core protein-protein interaction network.

Figure 6 The enriched GO items.

Figure 7 The enriched KEGG pathways.

Figure 8 The heatmap of docking scores of key targets combining to active compounds in JQP.

Figure 9 The representative docking complex of key targets and compounds of JQP in OA treatment, (A) AKT1 and puerarin; (B) AKT1 and salvianolic acid B; (C) TNF and salvianolic acid B.

Figure 10 RT-qPCR to examine HOTAIR expression (A); ELISA to test IL-1β, IL-6, IL-4, IL-10, APN levels (B-F). *** P < 0.001; ** P < 0.01; * P < 0.05.

Abbreviations: PBMCs, Peripheral Blood Mononuclear Cells; LncHOTAIR, Lnc RNA HOTAIR; APN, Adiponectin; IL-1β/4/6/10, Interleukin-1β/4/6/10.

Figure 11 RT-qPCR to check HOTAIR expression (A and B); CCK-8 assay to analyze PC viability (C); WB to test phosphorylation of PI3K and AKT and levels of APN (D); ELISA to determine IL-1β, IL-6, IL-4, IL-10, and APN levels (E–H); Semi-quantitative levels of APN, p-PI3K, and p-AKT detected with immunofluorescence (I–K). *** P < 0.001; ** P < 0.01; * P < 0.05.

Abbreviations: LncHOTAIR, Lnc RNA HOTAIR; PC, OA-PBMCs (Osteoarthritis-Peripheral Blood Mononuclear Cells) +OA-CH (Osteoarthritis-chondrocytes); APN, Adiponectin; IL-1β/4/6/10, Interleukin-1β/4/6/10.

Figure 12 CCK-8 assay to examine CH viability (A); Phosphorylation of PI3K and AKT and level of APN measured with WB (B); IL-1β, IL-6, IL-4, IL-10, and APN levels tested with ELISA (C–F); Semi-quantitative levels of APN, p-PI3K, and p-AKT checked with immunofluorescence (G–I). *** P < 0.001; ** P < 0.01.

Abbreviations: PC, OA-PBMCs (Osteoarthritis-Peripheral Blood Mononuclear Cells) +OA-CH (Osteoarthritis-chondrocytes); APN, Adiponectin; IL-1β/4/6/10, Interleukin-1β/4/6/10; ns, No statistical significance.

Figure 13 CH viability examined with CCK-8 assay (A); HOTAIR expression measured with PCR (B); Level of IL-1β, IL-6, IL-4, IL-10, and APN checked with ELISA (D-F); Phosphorylation of PI3K and AKT and level of APN tested with WB (G); Semi-quantitative levels of APN, p-PI3K, and p-AKT detected with immunofluorescence (H-J). ***P < 0.001; **P < 0.01; *P < 0.05.

Abbreviations: LncHOTAIR, Lnc RNA HOTAIR; PC, OA-PBMCs (Osteoarthritis-Peripheral Blood Mononuclear Cells) +OA-CH (Osteoarthritis-chondrocytes); APN, Adiponectin; IL-1β/4/6/10, Interleukin-1β/4/6/10.

Figure 14 CH viability measured with CCK-8 assay (A); HOTAIR levels tested with PCR (B); Phosphorylation of PI3K and AKT and level of APN checked with WB (C); IL-1β, IL-6, IL-4, IL-10, and APN levels examined with ELISA (D-G); Semi-quantitative levels of APN, p-PI3K, and p-AKT determined with immunofluorescence (H-J). *** P < 0.001; ** P < 0.01; * P < 0.05.

Abbreviations: JQP, Jianpi Qingre Tongluo Prescription; LncHOTAIR, Lnc RNA HOTAIR; PC, OA-PBMCs (Osteoarthritis-Peripheral Blood Mononuclear Cells) +OA-CH (Osteoarthritis-chondrocytes); APN, Adiponectin; IL-1β/4/6/10, Interleukin-1β/4/6/10; ns, No statistical significance.

Data Sharing Statement

The data that support the findings of this study are available from the information Center of Anhui Hospital of Traditional Chinese Medicine obtains the electronic medical record information of effective OA inpatients from the SQL Server database, but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are, however, available from the authors upon reasonable request and with permission of the information Center.