Jianwei Shoutai Pill reduces spontaneous abortion in mice by inhibiting Exo-miR-410-3p uptake and promoting trophoblast epithelial-mesenchymal transition, migration, and invasion

doi:10.12122/j.issn.1673-4254.2026.09.02

Abstract

Abstract:

Objective To investigate the therapeutic effect of Jianwei Shoutai Pill (JSP) on spontaneous abortion (SA) in mice and explore its therapeutic mechanism. Methods The miR-410-3p mimic was electroporated into milk exosomes to prepare exosomal miR-410-3p (Exo-miR-410-3p), which was characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. CCK-8, scratch wound healing, and Transwell assays were used to test the effects of control and JSP-medicated rat serum on proliferation, migration, and invasion of trophoblast HTR-8/Svneo cells. Female C57BL/6 mice were divided into SA model group, Model+Exo-miR-410-3p group, Model+Exo-miR-410-3p+P group, and Model+Exo-miR-410-3p+JSP group. The fluorescently labeled exosomes was tracked using in vivo and ex vivo imaging and embryo resorption rate was calculated. RT-qPCR, Western blotting, in situ hybridization, and immunohistochemical studies were carried out for further in vivo verification. Results Treatment with Exo-miR-410-3p significantly reduced migration and invasion capacities of HTR-8/Svneo cells, and these inhibitory effects were effectively reversed by JSP intervention. In pregnant C57BL/6 mouse models, JSP treatment prevented Exo-miR-410-3p from reaching the maternal-fetal interface. Consistently, the embryo resorption rate was significantly lower in Exo-miR-410-3p+JSP group than in Exo-miR-410-3p alone group [(20.73±3.77)% vs (74.95±10.09)%, P<0.01]. JSP treatment significant reduced miR-410-3p level and upregulated TRAF6 expression in the female mouse models. Conclusion JSP inhibits Exo-miR-410-3p uptake by trophoblast cells, thereby promoting trophoblast EMT, migration, and invasion and reducing embryo resorption in mouse models of spontaneous abortion.

Key words: Jianwei Shoutai Pill, spontaneous abortion, exosomal miR-410-3p, epithelial-mesenchymal transition, p38 MAPK signaling pathway

Zhanfa XU, Zhenyue CHEN, Liping XIE, Xinyi LI, Genggeng QIN, Jie GAO. Jianwei Shoutai Pill reduces spontaneous abortion in mice by inhibiting Exo-miR-410-3p uptake and promoting trophoblast epithelial-mesenchymal transition, migration, and invasion[J]. Journal of Southern Medical University, 2026, 46(9): 2006-2022.

Figures/Tables 15

Tab.1 Information of 3 constituent herbs in Jianwei Shoutai Pill (JSP)

Chinese name	Botanical^a name	English name	Genus family	Part uesd	Batch number	Weight (g)
Tusizi	Cuscuta chinensis Lam	Cuscutae semen	Convolvulaceae	Seed	150701	20
Sangjisheng	Taxillus sutchuenensis var.duclouxii (Lecomte) H.S.Kiu	Herba taxilli	Loranthaceae	Stem	160601	15
Xuduan	Dipsacus L.	Dipsaci radix	Caprifoliaceae	Stem	140705511	15

Fig.1 Chemoprofile and material basis of JSP lyophilized powder and JSP-medicated rat serum. A: Peak number and chemical characterization of JSP lyophilized powder in negative ion mode. B: Peak number and chemical characterization of JSP lyophilized powder in positive ion mode. C: Peak number and chemical characterization of JSP-containing serum in negative ion mode. D: Peak number and chemical characterization of JSP-containing serum in positive ion mode.

Tab.2 Top 10 ingredients of JSP lyophilized powder by HPLC-MS analysis under negative ion mode

No.	Identification	Formula	RT (min)	[M-H]^-(m/z)			MS2
No.	Identification	Formula	RT (min)	Measured	Predicted	Δ (ppm)	MS2
1	Phloroglucinol	C6H6O3	10.49	127.03892	126.03166	-0.304	109.02865, 99.04446
2	Mussaenosidic acid	C16H24O10	9.32	375.12921	376.13664	1.063	213.07642, 195.06569
3	Apocynin	C9H10O3	7.66	167.07027	166.06299	-0.025	167.07004, 152.04672
4	Methyl cinnamate	C10H10O2	10.91	180.10168	179.09449	-3.573	164.04254, 163.11104
5	6-O-Hexopyranosyl-1-O-[19-hydroxy -28-oxo-3-(pentopyranosyloxy)olean-12-	C47H76O18	16.94	973.5009	974.50825	0.493	928.50079, 927.49738
6	Salicylic acid	C7H6O3	12.96	137.02315	138.03042	-9.200	138.02663, 138.01686
7	Neochlorogenic acid	C16H18O9	8.18	353.08746	354.09470	-1.082	191.05544, 179.03421
8	4,5-Dicaffeoylquinic acid	C25H24O12	12.38	515.11902	516.12623	-1.051	354.09219, 353.08780
9	2-oxopiperidine-3-carbohydrazide	C6H11N3O2	15.25	156.08057	157.08784	-0.557	155.07317, 128.03448
10	Isorhamnetin	C16H12O7	13.65	317.06503	316.05776	-1.729	317.06497, 302.04166

Tab.3 Top 10 ingredients in JSP lyophilized powder by HPLC-MS analysis under positive ion mode

No.	Identification	Formula	RT (min)	[M-H]^-(m/z)			MS2
No.	Identification	Formula	RT (min)	Measured	Predicted	Δ (ppm)	MS2
1	Vanillin	C8H8O3	10.57	153.05446	152.04718	-1.066	136.07547, 135.11678
2	3-hydroxy-3-(2-pyridylmethyl)indolin-2-one	C14H12N2O2	9.59	241.09671	240.08944	-1.834	224.08940, 223.08618
3	Hyperoside	C21H20O12	12.85	465.10165	464.09481	-1.427	304.05289, 303.04953
4	α-Eleostearic acid	C18H30O2	21.85	279.23154	278.22430	-1.021	279.23132, 279.09326
5	Octyl decyl phthalate	C26H42O4	24.28	441.29675	440.28948	-1.272	315.15570, 203.17888
6	Betulin	C30H50O2	23.05	411.36124	410.35400	-1.438	397.26166, 359.24600
7	Maltol	C6H6O3	7.52	127.0390	126.03172	0.244	127.03897, 109.02870
8	6-O-[(2E)-3-(4-Hydroxyphenyl)-2-propenoyl]-1 -O-(3,4,5-trihydroxybenzoyl)hexopyranose	C22H22O12	11.73	477.10422	478.11149	0.961	477.10388, 433.11270
9	Quercetin	C15H10O7	13.57	303.04919	302.04192	-1.854	285.03885, 275.05548
10	Cafestol	C20H28O3	18.92	317.20819	316.20091	-1.739	299.19794, 271.20505

Tab.4 Top 10 ingredients of JSP-medicated rat serum by HPLC-MS analysis under negative ion mode

No.	Identification	Formula	RT(min)	[M-H]^-(m/z)			MS2
No.	Identification	Formula	RT(min)	Measured	Predicted	Δ(ppm)	MS2
1	Linoleic acid	C18H32O2	22.15	325.23828	280.24003	-0.715	279.23294, 59.01225
2	γ-Linolenic acid ethyl ester	C20H34O2	21.68	313.27289	312.26561	-1.761	257.24731, 239.23659
3	1-Stearoylglycerol	C21H42O4	23.04	381.29657	358.30739	-2.560	341.30453, 285.27875
4	(±)11(12)-EET	C20H32O3	19.75	319.22751	320.23479	1.717	301.21756, 257.22751
5	(±)13-HpODE	C18H32O4	19.39	295.22638	294.21925	-0.816	295.22440, 295.18799
6	4-Phenolsulfonic acid	C6H6O4S	10.45	172.99033	173.99760	3.218	93.03314, 79.95588
7	Deoxycholic Acid	C24H40O4	20.02	391.28513	392.29241	-0.648	365.30621, 347.29520
8	Progesterone	C21H30O2	16.98	315.23166	314.22438	-0.665	315.23148, 297.22113
9	4-Methylphenol	C7H8O	13.86	107.04849	108.05577	5.090	67.50848
10	5-trans-Prostaglandin E2	C20 H32O5	17.37	335.22116	334.21403	-2.094	335.22006, 317.21027

Tab.5 TOP 10 ingredients of JSP-medicated rat serum by HPLC-MS analysis under positive ion mode

No.	Identification	Formula	RT(min)	[M-H]^-(m/z)			MS2
No.	Identification	Formula	RT(min)	Measured	Predicted	Δ(ppm)	MS2
1	Adrenic acid	C22H36O2	23.00	331.26413	332.27136	-0.503	71.01227, 59.00959
2	Hexadecanamide	C16H3NO	21.56	256.26300	255.25574	-1.879	256.26297, 102.09174
3	Palmitoleic acid	C16H30O2	21.91	253.21693	254.22421	-1.453	59.01217, 53.94514
4	11(Z)-Eicosenoic acid	C20H38O2	24.23	309.28006	310.28733	0.489	309.17767, 104.95240
5	DL-Tryptophan	C11H12N2O2	7.28	188.07050	204.08978	-0.469	188.07042, 170.05983
6	Chenodeoxycholic Acid	C24H40O4	19.86	437.29034	438.29762	1.255	409.29636, 391.28537
7	Deoxycholic Acid	C24H40O4	18.52	391.28519	392.29249	-0.648	347.29453, 343.26413
8	Bis(4-ethylbenzylidene)sorbitol	C24H30O6	17.13	415.21078	414.20352	-1.736	135.08035, 119.08560
9	L-Phenylalanine	C9H11NO2	5.27	149.05977	148.05242	-5.921	131.04912, 120.08086
10	3-Methoxyphenylacetic acid	C9H10O3	11.28	165.05475	166.06213	-5.203	165.05470, 165.01831

Fig.2 Characterization and identification of Exo-Ctrl and Exo-miR-410-3p. A: TEM image of milk exosomes (Scale bar=200 nm). B: NTA of the size distribution of milk exosomes. C: Protein levels of CD63, CD81, and TSG101 in milk exosomes measured by Western blotting. D: Expression level of miR-410-3p in Exo-Ctrl or Exo-miR-410-3p measured by RT-qPCR. E: NTA of the size distribution of Exo-Ctrl or Exo-miR-410-3p. F: Fluorescence image of HTR-8/Svneo cells incubated with PKH26-labeled Exo-Ctrl or Exo-miR-410-3p (red) (Scale bar=25 µm). (n=3). ***P<0.001.

Fig.3 Exo-miR-410-3p suppresses EMT, migration, and invasion of HTR-8/Svneo cells. A: Expression level of miR-410-3p in HTR-8/Svneo cells treated with Exo-Ctrl or Exo-miR-410-3p measured by RT-qPCR. B, C: Protein levels of TRAF6, E-cadherin, N-cadherin, and vimentin in HTR-8/Svneo cells treated with Exo-Ctrl or Exo-miR-410-3p measured by Western blotting and immunofluorescence staining. D, E: Migration and invasion capacities of HTR-8/Svneo cells treated with Exo-Ctrl or Exo-miR-410-3p determined by wound healing and Transwell assays, respectively. Representative images of migrated or invaded cells are shown (Original magnification: ×200). (n=3). **P<0.01, ***P<0.001; ##P<0.01, ###P<0.001.

Fig.4 　JSP-medicated rat serum reversesinhibitory effects of Exo-miR-410-3p on HTR-8/Svneo cell EMT, migration, and invasion. A:Change of HTR-8/Svneo cell viability treatedwith Exo-miR-410-3p, control or JSPmedicatedserum for 48 h. B: Fluorescenceimage of HTR-8/Svneo cells incubated withPKH26-labeled Exo-miR-410-3p (red) treatedwith control or JSP-medicated serum (scalebar, 25 μm). C, D: Protein levels of TRAF6, Ecadherin,N-cadherin, and Vimentin in HTR-8/Svneo cells treated with control or JSPmedicatedserum under the action of ExomiR-410-3p measured by Western blottingand IF staining. E, F: Migration and invasioncapacities of HTR-8/Svneo cells treated withcontrol or JSP-containing serum under theaction of Exo-miR-410-3p determined bywound healing and Transwell assays,respectively. Representative images ofmigrated or invaded cells are shown (×200).(n=3). *P<0.05,**P<0.01, ***P<0.001, ##P<0.01 ###P<0.001

Fig.5 Effect of JSP-medicated serum on the p38 MAPK signaling pathway in HTR-8/Svneo cells. A, C: Protein levels of p38 and p-p38 in HTR-8/Svneo cells treated with Exo-Ctrl or Exo-miR-410-3p measured by Western blotting and IF staining. B, D: Protein levels of p38 and p-p38 in HTR-8/Svneo cells treated with control or JSP-medicated serum under the action of Exo-miR-410-3p measured by Western blotting and IF staining. (n=3). **P<0.01, ***P<0.001, ##P<0.01, ###P<0.001 vs indicated group.

Fig.6 The core role of the miR-410-3p/TRAF6/p38 MAPK pathway in JSP-mediated regulation. A, B: After transfection with miR-410-3p mimic or inhibitor in HTR-8/Svneo cells, the expression of TRAF6 and p-p38 proteins was detected by Western blotting. C: Effect of knocking down TRAF6 (si TRAF6) or overexpressing TRAF6 (LV TRAF6) on p-p38 protein expression. D: Rescue experiment of co transfection of miR-410-3p mimic and LV TRAF6. E, F: The p38 MAPK signaling pathway inhibitor SB203580 blocks the effects of miR-410-3p inhibitor or LV TRAF6. (n=3). *P<0.05, **P<0.01, ***P<0.001, #P<0.05, ##P<0.01, ###P<0.001.

Fig.7 Expression of E-cadherin, N-cadherin, and vimentin proteins under different conditions. A: After transfection of miR-410-3p mimic or inhibitor in HTR-8/Svneo cells, the protein expression of E-cadherin, N-cadherin, and vimentin was detected by Western blotting. B: Effect of knocking down TRAF6 (si TRAF6) or overexpressing TRAF6 (LV TRAF6) on the expression of E-cadherin, N-cadherin, and Vimentin proteins. C: Effect of co transfection of miR-410-3p mimic and LV TRAF6 on the expression of E-cadherin, N-cadherin, and vimentin proteins. (n=3). *P<0.05, **P<0.01, ***P<0.001, #P<0.05, ##P<0.01, ###P<0.001.

Fig.8 High doses of JSP reduces miR-410-3p in the serum, placenta, and uterus of female mice, but increases TRAF6 expression in the placenta. A-C: Level of miR-410-3p in the serum, placenta, and uterus of female mice treated with low, medium, and high doses of JSP detected by RT-qPCR (n=5). D: Expression level of TRAF6 protein in the placenta of female mice treated with low, medium, and high doses of JSP measured by Western blotting (n=3). *P<0.05, **P<0.01, ***P<0.001.

Fig.9 JSP can reduce embryo resorption rate induced by Exo-miR-410-3p. A: In vivo fluorescence images of DiR labeled Exo-miR-410-3p in female mice, representative ex vivo fluorescence images in different organs, and the quantification of ex vivo fluorescence images in embryos (n=5). B-D: Expression level of miR-410-3p in the serum, placenta, and uterus of female mice detected by RT-qPCR (n=5). E: Embryo resorption rate of female mice (n=5). F: Expression level of TRAF6 protein in the placenta of female mice measured by Western blotting (n=3). G, H: Distributions of miR-410-3p and TRAF6 in the placenta and uterus of female mice detected by ISH and IHC, respectively (Scale bars=50 μm, 25 μm). *P<0.05, ***P<0.001, #P<0.05, ##P<0.01, ###P<0.001.

Fig.10 Schematic diagram illustrating the mechanism by which JSP reduces SA by inhibiting Exo-miR-410-3p uptake to regulate trophoblast EMT, migration, and invasion.

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