Myrrh Essential Oil Improves DSS-Induced Colitis by Modulating the MAPK Signaling Pathway: In vitro and in vivo Studies

Figures & data

Table 1 Disease Activity Index (DAI) Score Table

Score	Weight Loss Rate (%)	Stool Consistency	Blood in Stool
0	0	Normal	Negative
1	1–5
2	6–10	Loose stool	Positive
3	11–15
4	15	Diarrhea	Rectal bleeding

Table 2 Qualitative Results of the Components in MEO by GC-MS

NO.	Library/ID	CAS	RI	Pct Total
1	delta-EIemene	20307–84-0	1337.92	0.996
2	Isolongifolene	1135–66-6	1389.59	6.598
3	beta-Elemene	515–13-9	1395.23	7.715
4	Longifolene	475–20-7	1406.41	0.219
5	gamma-Maaliene	20071–49-2	1419.55	0.690
6	Germacrene B	15423–57-1	1433.49	0.778
7	gamma-Selinene	515–17-3	1474.52	1.861
8	gamma-Muurolene	30021–74-0	1480.76	1.944
9	(-)-beta-Selinene	17066–67-0	1485.89	1.519
10	Curzerene	17910–09-7	1502.49	17.028
11	Eremophilene	10219–75-7	1506.70	1.097
12	(+)-delta-Cadinene	483–76-1	1523.24	0.390
13	(-)-alpha-Gurjunene	489–40-7	1555.85	5.202
14	Menthofuran	494–90-6	1578.94	0.843
15	Furanoeudesma 1.3-diene	87605–93-4	1577.92	21.984
16	Lindestrene	2221–88-7	1578.78	9.223
17	Atractylone	6989–21-5	1581.87	8.894
18	Germacrone	6902–91-6	1588.42	0.705
19	4,4’-Dimethoxybiphenyl	2132–80-1	1670.73	7.488
20	7-Acetoxy-p-mentha-1-ene-3-one	56248–42-1	1766.74	0.360
21	4-Methoxybenzhydrol	720–44-5	1773.14	2.382
22	1-Acetyl-4,6,8-trimethylazulene	834–97-9	1866.13	0.518
23	(4aS,10aS)-4a-Methyl-3,4,4a,9,10,10a-hexahydrophenanthren-1(2H)-one	62318–99-4	1952.31	0.752

Figure 1 GC-MS chromatogram of MEO and the structure of its components. (A) Total ion flow map of MEO. (B) Structures of 23 components in MEO.

Figure 2 The effect of MEO on cell viability in RAW264.7 macrophages. (A) Cell viability was measured by MTT cell viability assay. (B) Effect of MEO on NO production in LPS-induced RAW264.7 macrophages. (C) Effect of MEO on TNF-α production in RAW264.7. (D) Effect of MEO on IL-1β production in RAW264.7.

Notes: ^##P< 0.01 vs control group. **P< 0.01 vs model group.

Figure 3 The DAI, colon morphology and serum levels of TNF-α and IL-1β in colitis mice. (A) DAI scores. (B) Changes in the body weight in each group. (C) Morphological analysis of the colon in each group. (D) Differences in colon length in each mice group. (E) The levels of TNF-α in colon tissues of colitis mice. (F) The levels of IL-1β in colon tissues of colitis mice.

Notes: ^##P< 0.01 vs control group. *P< 0.05, **P< 0.01 vs model group.

Figure 4 Results of RNA-seq analysis in colitis mice with MEO. (A) PCA graph. (B) Volcano graph. (C) Heat map of DEGs. (D) KEGG enrichment results. (E) GO enrichment results.

Figure 5 Results of WGCNA. (A) Selection of the soft threshold value. (B) Gene clustering tree and module division. (C) Gene number distribution in each module. (D) Heat map for correlation analysis between two modules. (E) Heat map of gene expression and inter-module correlation analysis. (F) Results of correlation analysis of trait modules. (G) KEGG results enriched by the blue modules.

Figure 6 The results of MEO component–target network analysis and KEGG pathway enrichment analysis. (A) Intersection map of MEO targets, ulcerative colitis targets, and DEGs. (B) Component-target map of MEO. (C) PPI network map of the key targets. (D) KEGG enrichment analysis before introducing weighting coefficients. (E) KEGG enrichment analysis after introducing weighting coefficients.

Table 3 The Docking Scores of Key Targets with Active Components and Specific Ligands

Key Target	Active Components	Docking Score	Specific ligands	Docking Score
CACNA2D1	gamma-Muurolene	71.9261	Gabapentin	61.2152
CACNA2D1	Eremophilene	71.739
CACNA2D1	Germacrone	71.6017
CACNA2D1	4-Methoxybenzhydrol	71.5981
CACNA2D1	(+)-delta-Cadinene	71.5767
RAS	Curzerene	83.5249	Sotorasib	82.9067
RAS	Lindestrene	81.4724
RAS	Furanoeudesma 1.3-diene	80.6225
RAS	Germacrone	77.6854
RAS	(+)-delta-Cadinene	76.104
ERK	beta-EIemene	72.8193	Acetylsalicylic acid	58.4676
ERK	Curzerene	72.1743
ERK	Lindestrene	72.0177
ERK	gamma-Selinene	71.8568
ERK	delta-EIemene	71.8414
JNK	Furanoeudesma 1.3-diene	77.9532	Halicin	72.6008
JNK	4,4’-Dimethoxybiphenyl	76.2019
JNK	Lindestrene	73.3771
JNK	beta-Elemene	72.9658
JNK	Germacrone	71.7237

Figure 7 Molecular docking and thermogram results of the key targets with active components and specific ligands. (A) the docking results of 7mix protein of CACNA2D1 target with the specific ligand Gabapentin. (B) the docking results of 7mix protein of CACNA2D1 target with the gamma-Muurolene component. (C) the docking results of 3kkq protein of RAS target with the specific ligand Sotorasib. (D) the docking results of 3kkq protein of RAS target with the Curzerene component. (E) the docking results of 2ok1 protein of ERK target with the specific ligand Acetylsalicylic acid. (F) the docking results of 2ok1 protein of ERK target with the beta-Elemene component. (G) the docking results of 3v6r protein of JNK target with the specific ligand Halicin. (H) the docking results of 3v6r protein of JNK target with the Furanoeudesma 1.3-diene component. (I) the heat map of molecular docking.

Figure 8 The DAI score and colon morphology in colitis mice. (A) DAI scores. (B) Changes in the body weight of in each group. (C) Morphological analysis of the colon in different group. (D) Differences in colon length in each mice group. ^##P< 0.01 vs control group.

Note: **P< 0.01 vs model group.

Figure 9 The Results of MEO inhibition of serum inflammatory factor levels and effects on colonic histopathology in colitis mice. (A) Serum TNF-α levels of each mice group. (B) Serum IL-1β levels of each mice group. (C) Effect of MEO on DSS-induced histopathological changes in the colon. The yellow arrows show goblet cells in the normal colon, and the black arrow indicates inflammatory infiltration.

Note: **P< 0.01 vs model group, ^##P< 0.01 vs control group.

Figure 10 The Results Immunohistochemistry. (A) Effect of MEO on the levels of p-JNK, p-ERK, and TNF-α protein in DSS-induced colon tissues. (B) Protein levels of p-JNK in colon tissues of each mice group. (C) Protein levels of P-ERK in colon tissues of each mice group. (D) Protein levels of TNF-α in colon tissues of each mice group.

Note: ^##P< 0.01 vs control group, *P< 0.05, **P< 0.01 vs model group.

Figure 11 The results of Western blot. (A) Changes in the levels of P-JNK, JNK, P-ERK, and ERK in DSS-induced colon tissues after treatment with MEO. (B) Protein levels of p-JNK/JNK in colon tissues of each mice group. (C) Protein levels of p-ERK/ERK in colon tissues of each mice group.

Notes: ^##P< 0.01 vs control group. *P< 0.05 vs model group.

Figure 12 The mechanism of MEO in the treatment of colitis through the MAPK pathway.