Inhibitory effect of three components derived from Salvia miltiorrhiza against human cytomegalovirus

Abstract

Background

Human cytomegalovirus (HCMV) infection is responsible for congenital infections and poses a significant health risk to immunocompromised individuals. To date, there is no effective HCMV vaccine. Salvia miltiorrhiza is a well-known traditional Chinese herb that possesses active pharmacological effects including antiviral activity. However, no studies on the effects of Salvia miltiorrhiza on HCMV have been reported thus far.

Methods

Firstly, the cytotoxicity of five active components derived from Salvia miltiorrhiza was screened, then morphology, western blotting, qPCR, indirect immunofluorescence assay (IFA) and TCID₅₀ assay were used to assess anti-HCMV activity. Time-of-addition experiments were performed to identify the stage at which the active components were active. The mechanism of the anti-HCMV activity of the active component was also illustrated in this study.

Results

Salvianolic acid B, Tanshinone ⅡA, and Cryptotanshinone derived from Salvia miltiorrhiza were found to prevent lytic cytopathic changes, inhibit the expression of viral proteins, suppress the replication of HCMV DNA, and significantly reduce the viral titer in WI-38 cells. Time-of-addition experiments showed that they predominantly act during the Post-entry treatment (Post-T) stage. Moreover, they effectively suppressed HCMV-induced cellular senescence, which was evidenced by a decline in senescence-associated β-galactosidase activity, alleviated senescence-associated heterochromatin foci (SAHF), reduced expression of p16, p21, and p53, and decreased production of reactive oxygen species (ROS).

Conclusion

To our knowledge, this is the first report that salvianolic acid B, tanshinone IIA, and cryptotanshinone derived from Salvia miltiorrhiza exhibit anti-HCMV activity in vitro, and suggest potential for further development.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12985-026-03107-3.

Introduction

Human cytomegalovirus (HCMV) is a β-herpesvirus with ~ 240 kb double-stranded DNA (dsDNA) genome that encodes at least 165 proteins [1, 2]. The HCMV seroprevalence in adults is about 60% in developed countries and up to 90% in low- and middle- income countries [3]. HCMV shares numerous similar characteristics with other herpesviruses, including strict sequential gene expression and the lifelong presence in humans following primary infection [4]. HCMV infection is usually asymptomatic in people with healthy immune systems, but in immunocompromised individuals, it often leads to dangerous, life-threatening illnesses [5]. HCMV infection is an important contributor to microcephaly, cognitive impairment, visual impairment, sensorineural hearing loss, and vascular disease [6, 7]. Currently, there is no efficacious vaccine for HCMV infection [8, 9]. So for, Ganciclovir (GCV) and its prodrug Valganciclovir (VAV), Foscarnet (FOS), Cidofovir (CDV), Letermovir (LEV) and Maribavir (MAV) are approved antiviral agents for treatment or prevention of HCMV infection [5]. Ganciclovir and its prodrug Valganciclovir are the preferred antiviral drugs for the treatment of HCMV infection [5, 10]. Ganciclovir, Valganciclovir, Foscarnet and Cidofovir are similar in that they target the viral DNA polymerase and inhibit the synthesis of viral DNA [5, 10]. Letermovir targets the HCMV terminase complex and blocks the packaging of the viral DNA into capsids, which in turn prevents the formation of infectious virus particles [11]. Maribavir works by inhibiting the UL97 kinase enzyme and disrupts the assembly of the viral capsid [5, 12]. Although these drugs may have some therapeutic benefits, long-term use of the above drugs could lead to resistance and ultimately to treatment failure [13, 14]. Meanwhile, these drugs also induce side effects, including bone marrow suppression, nephrotoxicity, hepatotoxicity, electrolytes imbalance, carcinogenicity and reduced fertility [5].

Salvia miltiorrhiza (S. miltiorrhiza.), commonly referred as Danshen, is a well-known traditional Chinese herb that contains active ingredients such as diterpenoid quinones, hydrophilic phenolic acids, and essential oil constituents [15, 16]. It was first recorded in the Shenlong Bencao Jing (200–300 AD, Han Dynasty) which was the oldest medicine monograph in China [15, 17]. S. miltiorrhiza. has been used as not only in human medicine but also in health-promotion food [18]. S. miltiorrhiza. has been widely applied in the clinical treatment of cardiovascular and cerebrovascular diseases, diabetes, and circulatory system diseases [17, 19, 20]. Furthermore, S. miltiorrhiza. possesses antioxidant [21], anti-inflammatory [22], antibacterial [23], and anti-tumor properties [24]. Meanwhile, extracts of S. miltiorrhiza. have been reported to exert antiviral activity, such as SARS-CoV-2 [25–28], HIV [29–32], HBV [33–35], enterovirus 71 [36–40]. Promoting the modernization of traditional Chinese medicine (TCM) requires not only the study of the molecular mechanisms by which TCM works but also the continuous development of new uses and functions of TCM. Based on the fact that S. miltiorrhiza. has been shown to possess such a rich variety of biological activities, we wondered whether S. miltiorrhiza. also possesses anti-HCMV effects. In this study, we report for the first time the anti-HCMV effects of several major active ingredients in S. miltiorrhiza. in vitro.

Materials and methods

Reagents, cells and virus

Salvianic acid A sodium (SAAS, 67920-52−9), Tanshinone I (TS I, 568-73−0), Salvianolic acid B (SAB, 121521-90−2), Tanshinone ⅡA (TS ⅡA, 568-72−9), and Cryptotanshinone (CTS,35825-57−1) (Fig. 1), five compounds of S. miltiorrhiza. were purchased from Shanghai Standard Technology Co., Ltd. (Shanghai, China) and dissolved in Vehicle (DMSO) for a stock solution of 20 mM. FOS (SC-205330) was obtained from Santa Cruz Biotechnology. Human diploid fibroblasts (WI-38 cells) and HCMV (Towne strain) were obtained from the American Tissue Culture Collection (Manassas, VA, USA). As we demonstrated previously [41, 42], WI-38 cells were considered to be young when the population doubling (PD) number was 30 or less and replicative senescence at PD ≥ 50. All experiments in this study were performed with young cells in the range of 25 to 30 PD.

Fig. 1.

Chemical structure of SAAS, SAB, TS I, TS ⅡA, and CTS derived from S. miltiorrhiza

Cell cytotoxicity

Cell cytotoxicity analysis of these compounds used in the study was performed using CCK-8 Assay Kit (RM02823, Abclonal Technology, Wuhan, China) as described previously [43]. Firstly, WI-38 cells were seeded in 96-well plates at 5 × 10³ cells/well in DMEM with 10% FBS. The following day, when the cells grew into monolayers and were washed with PBS. Secondly, Culture medium with 0.2% FBS containing different drug concentrations (0 µM, 1 µM, 2 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM, 300 µM) was then added and incubated for another 5 days. Finally, 10 µL CCK-8 was applied to each well for 1–2 h at 37 °C, and the absorbance was measured using a Thermo Scientific microplate reader (Multiskan™ FC) at 450 nm following the standard protocol. CC₅₀ (half cytotoxic concentration) was calculated using the above data. Each group was assessed in Sextuplicate.

Antiviral activity of S. miltiorrhiza. compounds

HCMV infection and S. miltiorrhiza. compounds treatment

Stocks of the Towne strain of HCMV were routinely prepared as previously mentioned [41–44]. WI-38 cells were cultured in DMEM (GIBCO) with 10% fetal bovine serum (FBS, GIBCO) and 100 × penicillin-streptomycin solution (GIBCO); then, WI-38 cells were synchronized in the G0/G1 phase by replacing the culture medium with 0.2% FBS for 48 h before HCMV infection. The synchronized WI-38 cells were mock- infected or infected with HCMV at the multiplicity of infection (MOI) of 0.5 for 1 h at 37℃. The cells were harvested at the indicated time points post-infection. FOS (200 µg/ml) was set as positive-drug control. In the FOS and S. miltiorrhiza. compounds treatment group, the synchronized WI-38 cells were treated with S. miltiorrhiza. compounds 2 h before HCMV infection.

Western blotting

The cell samples were prepared as described above and lysed with cell lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Then, 50 µg of protein was subjected to SDS-PAGE for separation and transferred onto PVDF membranes (Cell Signaling Technology, MA, USA). Next, the membranes were blocked with 5% non-fat dry milk for 1 h at room temperature, and incubated with primary antibodies of anti-IE1/2 (P1215, Virusys Corporation, Randallstown, USA), anti-p52 (CA006-100, Virusys Corporation, Randallstown, USA), anti-p16 (ET1608-62, Huaan Biotechnology, Hangzhou, China), anti-p21 (2947 S, Cell Signaling Technology Boston, USA), anti-p53 (2527 S, Cell Signaling Technology Boston, USA) and anti-GAPDH (AC033, Abclonal Technology, Wuhan, China). After incubation with secondary antibody linked to HRP, the immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) method (RM02867, Abclonal Technology, Wuhan, China).

qPCR

The cell samples were prepared as described above. Viral DNA was obtained using QIAamp DNA Mini Kit (Hilden, Germany) according to the manufacturer’s instructions. The copy number of HCMV DNA was evaluated using qPCR assay with 2 × Universal SYBR Green Fast qPCR Mix (Abclonal Technology, Wuhan, China) in a reaction volume of 20 µl. The HCMV UL123, UL44, UL32 and GAPDH primers used in this study were listed in Table 1. The qPCR protocol was as shown below: 3 min at 95 °C for initial denaturation, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. Each group was tested in triplicate (n = 3). The difference in HCMV DNA copy number of UL123, UL44 and UL32 was calculated using the 2^−△△Ct method normalized to GAPDH as previously mentioned [43].

Table 1.

Primer sequences used in this study

Gene name	Primer name	Sequence
UL123	UL123-forward UL123-reverse	5'-TCTGCCAGGACATCTTTCTC-3' 5′-GTGACCAAGGCCACGACGTT-3'
UL44	UL44-forward UL44-reverse	5'-ACTGCCGTGCACGTTGCGTA-3' 5'-ACTTGCCGCTGTTCCCGACG-3'
UL32	UL32-forward UL32-reverse	5/- GGTTTCTGGCTCGTGGATGTCG-3' 5'- CACACAACACCGTCGTCCGATTAC-3'
GAPDH	GAPDH -forward GAPDH -reverse	5'-CTGTTGCTGTAGCCAAATTCGT-3' 5'-ACCCACTCCTCCACCTTTGAC-3'

Indirect immunofluorescence assay (IFA)

IFA was conducted in a 12-well plate, and the drug treatment and HCMV infection were performed as above. The cell samples were collected at 5 dpi and fixed for 15 min at room temperature using a 4% paraformaldehyde. Subsequently, they were blocked with 5% non-fat dry milk for 1 h at room temperature. After washing thrice with PBS, the cell samples were exposed to mouse monoclonal antibody to anti-HCMV IE1/2 overnight at 4 °C and washed thrice with PBS, followed by incubation with FITC-conjugated goat anti-mouse IgG (HA1003, Huaan Biotechnology, Hangzhou, China) and washed thrice with PBS. Cell nucleus were stained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime Institute of Biotechnology, Shanghai, China) for 15 min at room temperature and washed thrice with PBS. Finally, the IE1/2 positive fluorescence signal was observed using a Zeiss microscope (Axio Vert.A1).

Tissue culture infective dose (TCID₅₀) assay

The TCID₅₀ assay was carried out to investigate viral titer in a 96-well plate and the method was described as previous study [42–44]. Briefly, WI-38 cells were seeded in a 96-well plate one day ahead, when the cells reached to 80% confluence. The different HCMV samples were serially diluted tenfold containing 0.2% FBS. Subsequently, the culture medium was discussed and an aliquot of 100 µL from each dilution was inoculated into WI-38 cells and maintained at 37 °C in a 5% CO₂ incubator. The experiment was performed simultaneously in 8 replicates per dilution. Cytopathic effects (CPE) changes were monitored and recorded every day until the number of wells showing CPE changes did not increase. Finally, the virus titer was calculated according to the classic Reed-Muench method [45].

Time‑of‑addition experiments

The time-of-addition assay was performed under five different experimental conditions depending on when the drug was added. HCMV infection and drug treatment were terminated by discarding the culture medium, washing WI-38 cells with PBS and replacing fresh medium. Group 1 was named Full time treatment (Full time-T), drug was diluted in fresh culture medium with 0.2% FBS and added to the WI-38 cells for 2 h (−2 h) before viral inoculation, in this group, the drug was always present in the WI-38 cells throughout the experiment. Group 2 was named Pre-treatment cell (Pre cell-T), drug was applied to WI-38 cells for 2 h ahead of infection. Group 3 was named Co-treatment (Co-T), drug and HCMV were simultaneously added to the WI-38 cells, ensuring that the drug was present during the infection process. Group 4 was named Pre-treatment virus (Pre virus-T), drug pretreated with HCMV for 2 h at 4℃ and the HCMV- drug mixture was added to WI-38 cells for another 2 h incubation. Group 5 was named Post-entry treatment (Post-T), the viral inoculum was added to WI-38 cells and incubated for 2 h. Then, we removed the viral inoculum and washed the cells thrice with preheated PBS. Finally, fresh culture medium with 0.2% FBS with or without drug was added to corresponding well and maintained until sample collection. The cell samples of each group were collected at 72 hpi for Western blot analysis and qPCR assay. The concentration of SAB, TS ⅡA, CTS was 10 µM, and the MOI of HCMV was 5. The cell samples of each group were collected at 72 hpi for Western blot analysis and qPCR assay.

Senescence‑associated beta‑galactosidase (SA‑β‑gal) staining

SA-β-gal activity was evaluated with a SA-β-galactosidase staining kit (C0602, Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer’s protocol. Briefly, The WI-38 cells were seeded in 12-well plates one day ahead. Then, the cells were subjected to various drugs treatment and HCMV infection at 5 dpi. Subsequently, the cells were washed thrice with PBS and fixed for 15 min. After washing with PBS, cells were stained with freshly prepared β‑gal detection solution at 37 °C overnight, and paid more attention to evaporation in this step. Finally, the cells were washed thrice with PBS and overlaid with PBS. Images were captured randomly using a Zeiss microscope (Axio Vert.A1). SA-β-gal-positive cells (blue-green cells) were indicated for cellular senescence, and the percentage of senescent cells was assessed based on three independent experiments.

Measurement of intracellular reactive oxygen species (ROS)

Intracellular ROS level was evaluated with ROS Assay Kit (S0033S, Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, the cells were seeded in 6-well plates, and the cell samples were treated as above. the ROS probe 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFDA) was diluted in serum-free DMEM at 1:1000 to a final concentration of 10 µM. Then, the cells were washed thrice with serum-free DMEM and incubated with 1 ml H₂DCFDA for 20–30 min at 37 °C. Next, the cells were washed thrice with serum-free DMEM to fully remove H₂DCFDA which did not enter the cells. Finally, the cells were observed using a Zeiss microscope (Axio Vert.A1) and harvested using 0.25% trypsin-EDTA (Gibco, NY, USA). The intracellular fluorescence intensity was evaluated by flow cytometry.

Statistical analysis

Data are presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to determine significant differences between the various groups. Statistical significance was set at P < 0.05.

Results

Effect of S. miltiorrhiza. compounds on WI-38 cell viability

We investigated the cell viability of S. miltiorrhiza. compounds on WI-38 cells using CCK-8 Assay Kit. No significant cytotoxic effects were observed when WI-38 cells were exposed to concentrations ranging from 1 to 10 µM for 5 days (P > 0.05).The half cytotoxic concentration (CC₅₀) in WI-38 cells of SAAS, TS I, SAB, TS ⅡA, and CTS was summarized in Table 2 respectively. SAAS showed the highest CC₅₀ (Table 2). Therefore, we choose a concentration of no more than 10 µM as the concentration for subsequent studies.

Table 2.

Antiviral activity of SAAS, TS I, SAB, TS ⅡA, and CTS on HCMV

Compound	CC50 (µM)a	EC50 (µM)b	EC90 (µM)c	SId
SAAS	143.3±7.2	n.d. e	n.d.	n.d.
TS I	18.4±0.1	n.d.	n.d.	n.d.
SAB	57.8±2.9	8.2±0.2	19.1±1.2	7
TS ⅡA	36.2±6.9	5.1±0.2	9.4±2.1	7
CTS	46.3±5.1	1.6±0.4	7.2±1.2	29

^aCC₅₀, compound concentration producing 50% cytotoxicity, as determined by CCK-8 Assays performed in WI-38 cells. Reported values are the Means ± SD of data derived from nine experiments performed in Sextuplicate

^b EC₅₀, the compound concentration inhibiting 50% of virus titer, as determined according to the classic Reed-Muench method with WI-38 cells. Reported values are the Means ± SD of data derived from eight experiments performed in triplicate

^cEC₉₀, the compound concentration inhibiting 90% of virus titer, as determined according to the classic Reed-Muench method with WI-38 cells. Reported values are the Means ± SD of data derived from eight experiments performed in triplicate

^dSI, Selectivity Index determined as the ratio of CC₅₀ to EC₅₀

^en.d, indicates that the compound was not tested in that specific assay

Anti-HCMV activity of SAAS, TSI, SAB, TS IIA, and CTS

Since our group previously observed morphological changes and CPE in HCMV- infected cells [42–44]. As shown in Fig. 2A, we could obviously observe the CPE after 5 days of HCMV infection alone in bright field image (BF). Among S. miltiorrhiza. compounds pretreated groups, SAAS and TS I (10 µM) pretreatment showed typical CPE similar to that of HCMV infection alone, we were surprised to find that the degree of CPE was significantly relieved in wells treated with 10 µM SAB, TS IIA, and CTS. The IFA result showed that IE1/2 positive signal decreased extremely when the WI-38 cells were pretreated with SAB, TS IIA, and CTS (10 µM) (Fig. 2A and B). The results above manifested that SAB, TS IIA, and CTS could protect the morphological feature of WI-38 cells infected with HCMV and suggested that these three compounds might have anti-HCMV activity. To further confirm the anti-HCMV effect of SAB, TS ⅡA, and CTS, we performed Western blotting and qPCR to assess the viral protein expression and viral gene copy number, respectively. The Western blotting results were shown in Fig. 2C and D, HCMV immediate early protein IE1/2 and early protein p52 were both reduced rapidly compared to HCMV infection alone, and the downregulation effect of SAB and CTS was more siginificant, which was more than 50%. qPCR data was also consistent with the result in protein levels, SAB, TS ⅡA, and CTS reduced the gene copy number of the immediate early gene UL123, the early gene UL44, and the late gene UL32 (Fig. 2E). These results suggest that the three compounds have good anti-HCMV activity.

Fig. 2.

Antiviral activity evaluation of SAAS, SAB, TS I, TS ⅡA, and CTS on HCMV. WI-38 cells were pretreated with or without 10 µM SAAS, SAB, TS I, TS ⅡA, and CTS 2 h, respectively, before HCMV infection (MOI = 0.5) and collected at indicated time to subject to different evaluation methods. (A) and (B) IFA was performed at 5 dpi. The IE1/2-positive signals were detected by a mouse IE1/2 monoclonal antibody combined with FITC-conjugated goat anti-mouse IgG. Representative images were acquired and shown here. Bar: 100 μm. (C) and (D) Expression levels of IE1/2 and p52 were determined at 5 dpi by Western blotting. GAPDH was used as the loading control. Quantitative analysis of protein levels from three independent experiments. (E) The copy numbers of UL123, UL44, and UL32 were measured with the 2^−△△Ct method. The HCMV DNA was extracted at 3 dpi as mentioned in the Materials and Methods section. The group of HCMV alone was used as control. *P < 0.05, ***P < 0.001 VS the control

Finally, we studied the anti-HCMV activity of three compounds at different concentrations. We performed Western blotting, qPCR, and TCID₅₀ assay to assess the viral protein expression, viral gene copy number and virus titer when WI-38 cells were pretreated with three different concentrations (1 µM, 5 µM, 10 µM), respectively. For SAB and CTS, when the concentration reached to 5 µM or higher, both the viral protein level of IE1/2 and p52, and the gene copy number of UL123, UL44 and UL32 decreased rapidly (Fig. 3A, B, C, G, H and I). For TS ⅡA, the anti-HCMV activity was weaker than that of the other two, and only when the concentration reached to 10 µM could it showed anti-HCMV activity at the protein level (P < 0.001) (Fig. 3D and E). The viral gene copy unmber of UL123 and UL44 could be significantly reduced at a concentration of 5 µM (P < 0.05), while the copy number of UL32 had no significant difference at this concentration, and only when the concentration reached to 10 µM, there was a significant difference (Fig. 3F). Representative cell morphology images were shown in Fig. S1. The TCID₅₀ assay was performed to examine the changes in viral titer caused by SAB, TS ⅡA, and CTS treatment, respectively. The results were shown in Table 2, and the half-maximal effective concentration (EC₅₀) of SAB, TS ⅡA, and CTS was determined to be 8.2 ± 0.2 µM, 5.1 ± 0.2 µM, 1.6 ± 0.4 µM, respectively. Meanwhile, EC₉₀ of SAB, TS ⅡA, and CTS was determined to be 19.1 ± 1.2 µM, 9.4 ± 2.1 µM, 7.2 ± 1.2 µM, respectively.Therefore, the selectivity index (SI) of them was calculated as 7, 7 and 29 reepectively. CTS possessed the best inhibitory effect among them.

Fig. 3.

Antiviral activity of SAB, TS ⅡA, and CTS on HCMV. WI-38 cells were pretreated with or without different concentrations of SAB, TS ⅡA, and CTS (1 µM, 5 µM, 10 µM) for 2 h, respectively, before HCMV infection (MOI = 0.5) and collected at indicated time. The anti-HCMV activity was assayed through Western bloting, qPCR and TCID₅₀ methods. (A) and (B) Representative images and quantitative analysis of expression levels of IE1/2 and p52 for SAB treatment. (C) The copy numbers of UL123, UL44, and UL32 for SAB treatment. (D) and (E) Representative images and quantitative analysis of expression levels of IE1/2 and p52 for TS ⅡA treatment. (F) The copy numbers of UL123, UL44, and UL32 for TS ⅡA treatment. (G) and (H) Representative images and quantitative analysis of expression levels of IE1/2 and p52 for CTS treatment. (I) The copy numbers of UL123, UL44, and UL32 for CTS treatment. (J-L) Effect of SAB, TS ⅡA, and CTS treatment on virus titer by TCID₅₀ assay. WI-38 cells infected with HCMV (MOI = 0.5) with or without SAB, TS ⅡA, and CTS treatment (0.01 µM, 0.1 µM, 1 µM, 2 µM, 5 µM, 10 µM) for 5 dpi, respectively. The cell samples were collected and freeze-thawed thrice. The virus titer was calculated according to Reed-Muench method. The half-maximal effective concentration (EC50) was calculated by TCID₅₀ results. The group of HCMV alone was used as control. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control

SAB, TSⅡA, and CTS suppressed HCMV at the post‑entry stage

Time-of-addition assay was performed to figure out in which step SAB, TS ⅡA, and CTS exerted their antiviral activity. Pretreatment of SAB, TS ⅡA, CTS and HCMV infection were applied to the WI-38 cells as described in the section of Materials and Methods and summarized in Fig. 4A. We analyzed the IE1/2 viral protein expression level (Fig. 4B − 4G) and the gene copy number of UL123 (Fig. 4H) in five different treatment groups. Compared to the Control group, drugs treatment throughout the experiment (Group 1) significantly reduced the expression level of IE1/2 which also verified the anti-HCMV activity of SAB, TS ⅡA, CTS. The expression level of IE1/2 did not change significantly in the “Pre cell-T” (Group 2), “Co-T” (Group 3), “Pre virus-T” group (Group 4) (P > 0.05), but in the “Post-T” (Group 5) assay, the changes were significantly different (Fig. 4B - G). The result showed that SAB, TS ⅡA, and CTS suppressed HCMV at the “Post-T” stage, and did not affect the early stages of the virus’s life cycle, such as its attachment to and entry into cells (Group 2). Meanwhile, SAB, TS ⅡA, and CTS also did not demonstrate obvious virucidal ability in vitro (Group 4). The result of the gene copy number of UL123 was consistent with those of the protein above ((Fig. 4H).

Fig. 4.

SAB, TS ⅡA, and CTS exert anti-HCMV effects at the Post-Entry stage. (A) Schematic representation of “Full time-T” (Group 1),“Pre cell-T” (Group 2), “Co-T” (Group 3), “Pre virus-T” (Group 4), “Post-T” (Group 5) experimental design. In Group 1, SAB, TS ⅡA, and CTS (10 µM), repectively, was applied to the cells 2 h prior to HCMV infection (MOI = 5) and treated the cells throughout the experiment. In Group 2, SAB, TS ⅡA, and CTS (10 µM), repectively, pretreated with the cells for 2 h and was removed before HCMV infection (MOI = 5). In Group 3, SAB, TS ⅡA, and CTS (10 µM), repectively, was applied to the cells during the HCMV infection (MOI = 5) for 2 h. In Group 4, SAB, TS ⅡA, and CTS (10 µM), repectively, pretreated with HCMV for 2 h in 4℃ before the HCMV- drug mixture was incubated with the cells for another 2 h. In Group 5, WI-38 cells were infected with HCMV (MOI = 5) for adsorption, entry for 2 h. After washing thrice with PBS, the fresh culture medium containing SAB, TS ⅡA, and CTS (10 µM), repectively, was added to WI-38 cells for another 72 hpi for western blotting assay. (B) (D) (F) Representative images of the expression level of viral protein IE1/2 for “Time-of-addition” treatment with SAB, TS ⅡA, and CTS, respectively. (C) (E) (G) Quantitative analysis of the expression level of viral protein IE1/2 for “Time-of-addition” treatment with SAB, TS ⅡA, and CTS, respectively. (H) The copy numbers of UL123 for “Time-of-addition” treatment with SAB, TS ⅡA, and CTS, respectively. The group of HCMV alone (+) was used as control. *P < 0.05, **P < 0.01, ***P < 0.001 VS the group of control

SAB, TS ⅡA, and CTS suppressed HCMV-induced cellular senescence

Previous reports indicated that HCMV infection could induce cellular senescence in WI-38 cells, which were also widely used as cellular senescence model [41–43, 46, 47]. The most intuitive and precise cell phenotype was the accumulation of a lysosomal enzyme named SA-β-gal in cultured cells, so we performed the classical SA-β-gal staining to detect the cellular senescence level in young WI-38 cells (PD 30) subjected to HCMV infection with or without drugs treatment. As demonstrated in Fig. 5A and B, young WI-38 cells (Mock, PD 30) displayed a limited number of positive cells (blue-green). In contrast, the HCMV-infected groups (HCMV alone or HCMV+Vehicle) could induce about 70% cellular senescence at 5 dpi, which was consistent with prevous studies [42, 43]. While the percentage of SA-β-gal positive cells decreased to 40% when treated with 10 µM TS ⅡA, and there was a significant difference compared with HCMV alone group (P < 0.001). More visibly, the percentage of SA-β-gal positive cells dramatically reduced to about 10% when treated with SAB and CTS, respectively, it was worth noting that the effect was similar with the Mock group (P > 0.05). The result indicated that SAB, TS ⅡA, and CTS possessed significant inhibitory effect on cellular senescence triggered by HCMV infection.

Fig. 5.

Effect of SAB, TS ⅡA, and CTS on SA-β-gal staining and SAHF in HCMV-infected WI-38 cells. (A) Effect of SAB, TS ⅡA, and CTS on SA-β-gal staining in HCMV-infected WI-38 cells. WI-38 cells were synchronized in the G0/G1 phase by replacing the culture medium with 0.2% FBS for 48 h and pretreated with or without SAB, TS ⅡA, and CTS 2 h before HCMV inoculation (MOI = 0.5). After 5 dpi, WI-38 cells were submitted for SA-β-gal staining according to the Kit instruction. Representative images were shown here from three independent experiments. Bar: 100 μm. (B) Quantitative analysis of SA-β-gal positive cells from three independent experiments. ^###P < 0.001 VS mock. ***P < 0.05 VS HCMV alone group. (C) Effect of SAB, TS ⅡA, and CTS on SAHF in HCMV-infected WI-38 cells. WI-38 cells were pretreated with or without SAB, TS ⅡA, and CTS 2 h before HCMV inoculation (MOI = 0.5). After 5 dpi, WI-38 cells were washed with PBS and fixed with 4.0% paraformaldehyde, and the cell nucleus were stained by DAPI for 15 min at room temperature and washed thrice with PBS. Finally, the SAHF signal was observed using a Zeiss microscope (n = 3). Bar: 20 μm

At the chromatin level, alterations in heterochromatin structure were thought to contribute to the maintenance of the irreversible state of cellular senescence. Characteristic heterochromatin structures called senescence-associated heterochromatin foci (SAHF), with dot-like aggregation, were formed in senescent cells, which was used as a marker of senescent cells [48, 49]. In HCMV infection alone or with Vehicle groups, we could observe SAHF phenomenon obviously compared with Mock group, indicating that HCMV infection induced the formation of SAHF, but in the SAB, TS ⅡA, and CTS treatment groups, the phenomenon was significantly alleviated (Fig. 5C). In conclusion, we believed that SAB, TS ⅡA, and CTS could effectively suppressed HCMV-induced cellular senescence.

SAB, TSⅡA, and CTS suppressed HCMV‑induced activation of molecular mechanisms of cellular senescence

p53/p21 and p16/Rb pathways played important roles in the initiation of cellular senescence and could also be used as biomarkers of cellular senescence [48–50]. As shown in Fig. 6, the Vehicle alone did not change the expression level of p53, p21 and p16 (P > 0.05), while HCMV infection significantly upregulated these proteins expression levels (P < 0.001), and the result was in accordance with previous studies [42, 43]. However, SAB, TS ⅡA, and CTS treatment alleviated the effect. The results indicated that SAB, TS ⅡA, and CTS could significantly inhibit the increase of p16, p21, and p53 induced by HCMV infection.

Fig. 6.

Effect of SAB, TS ⅡA, and CTS on HCMV-induced expression of p16, p21, and p53. (A) WI-38 cells were serum starved and synchronized in the G0/G1 phase. Then, WI-38 cells were pretreated with SAB, TS ⅡA, and CTS (10 µM) for 2 h and followed by HCMV infection (MOI = 0.5) for 5 dpi, and then, the cell samples were harvested to subject Western blotting to detect the expression level of p16, p21, and p53. GAPDH was used as the loading control. Representative images shown here were selected from three independent experiments. (B) Quantitative analysis of the three protein levels. The cell sample group without HCMV infection was set as mock group, ^#P < 0.05 VS mock, ^###P < 0.001 VS mock. *P < 0.05 VS HCMV alone group, **P < 0.01 VS HCMV alone group, ***P < 0.001 VS HCMV alone group

SAB, TSⅡA, and CTS suppressed HCMV‑induced ROS production

Accumulation of oxidative stress was related to the progress of cellular senescence and virus infection [51–53], so we measured intracellular ROS level in HCMV-infected WI-38 cells with or without SAB, TS ⅡA, and CTS treatment. In this study, the change pattern of ROS production was consistent with previous studies, which was that HCMV infection alone could cause a sharp increase, while drug treatment could significantly reduce the ROS increase caused by viral infection [41–43] (Fig. 7). These findings proved that SAB, TS ⅡA, and CTS could efficiently alleviate HCMV-induced ROS generation.

Fig. 7.

Effect of SAB, TS ⅡA, and CTS on HCMV-induced ROS production. WI-38 cells were serum starved and synchronized in the G0/G1 phase. Then, WI-38 cells were infected with HCMV (MOI = 0.5) with or without treatment of SAB, TS ⅡA, and CTS (10 µM) for 5 dpi. And the cell samples were incubated with the probe H₂DCFDA. (A) Representative images were observed using a Zeiss microscope before submitting to Flow cytometry. (B) Quantitative analysis of the ROS levels obtained from the data of three independent experiments. WI-38 cells untreated group was set as mock, HCMV+Vehicle group was used as control. ^##P < 0.01 VS mock, ^###P < 0.001 VS mock. ***P < 0.001 VS HCMV+Vehicle group. Bar: 100 μm

Discussion

The continued emergence of drug-resistant strains and side effects of medication are major challenges in HCMV treatment. For this reason, it is strongly necessary to develop new alternative drugs for HCMV infection, and drug repurposing is one of the promising, convenient and cost- and time-effective methods. The active compounds of S. miltiorrhiza can be divided into two major groups: lipophilic tanshinones including tanshinone I (TS I), tanshinone IIA (TS ⅡA), dihydrotanshinone (DTS), and cryptotanshinone (CTS) [17, 54], and hydrophilic phenolic acids such as salvianolic acid A (SAA), salvianolic acid B (SAB) [18]. In this study, we select five of them (SAAS, SAB, TS I, TS Ⅱ A, and CTS) to evaluate their anti-HCMV activities and provide a new cue of anti-HCMV compounds derived from S. miltiorrhiza.

Compounds derived from S. miltiorrhiza have been found to possess a wide range of pharmacological activities, such as antibacterial, antiviral, antioxidant, anti-inflammatory, and anti-tumor properties. As for antiviral activity, current researches mainly focus on HIV [29–31], SARS-CoV-2 [25–28], HBV [33–35], and EV71 [36–40]. Different from the other virus, HCMV genome is expressed in a temporally coordinated and regulated cascade of transcriptional events, and the genes are divided into three categories in order of precedence, designated immendiately early (IE), early (E), and late (L) [55]. The IE gene plays a crucial role in the expression and regulation of downstrean gene [56], while E gene is necessary for viral DNA replication [57], the L gene encoding proteins mainly structural roles and primarily contribute to the assembly and morphogenesis of the virion [55]. Therefore, we choose the expression level of IE1/2 (IE gene product), p52 (E gene product) proteins and the copy number of UL123, UL44, and UL32 gene as representations of the three sequential gene stages to evaluate the anti-HCMV activity of five compounds form S. miltiorrhiza in this study. The result reveals that SAB, TS ⅡA, and CTS instead of SAAS and TS I pretreatment groups could downregulate all of the indicators mentioned above and demonstrate their anti-HCMV activities (Figs. 2 and 3).

Although replication of different viruses varies and has specific characteristics, they share several common stages during the viral life cycle, including adsorption, entry, biosynthesis, assembly, maturation, and egress, which are all possible targets [58]. Therefore, identifying the specific stage of drug action is crucial to the further study of antiviral mechanisms. As shown in Fig. 4, the result of Time-of-addition demonstrates that SAB, TS ⅡA, and CTS play an antiviral role in the Post-T stage (Group 5) both in the viral proteins and gene copy number level. It is worth noting that pretreatment of SAB, TS ⅡA, and CTS 2 h with WI-38 cells (Pre cell-T, Group 2) and HCMV (Pre virus-T, Group 4) before HCMV inoculation does not result in reduction of viral proteins expression and gene copy number. This result indicates that SAB, TS ⅡA, and CTS do not affect the stages of HCMV adsorption and entry, nor does it inactivate the virus in vitro.

Cellular senescence is a response elicited by acute or chronic damage, including oncogenic signaling, mitochondrial damage, telomere shortening, genotoxic damage, oxidative damage, mechanical stress, nutrient imbalance, and virus or bacteria infection [59–61]. Virus-induced senescence (VIS) has attracted more attention and an increasing number of studies are emerged, including SARS-CoV-2 [62–64], influenza A Virus [65], measles virus [66], human respiratory syncytial virus [67], endogenous retroviruses [68]. HCMV, like other viruses, can cause cellular senescence and exacerbates the senescence-associated secretory phenotype (SASP), which is mainly composed of comprised largely of pro-inflammatory, extracellular matrix-degrading, complement-activating and pro-coagulatory factors secreted by senescent cells. These effects are enhanced in elderly individuals who have an increased proportion of pre-existing senescent cells in their bodies [64]. Latent infection is a significant characteristic of HCMV. After the initial infection with HCMV, it will remain latent within the body, and the latent period is variable. HCMV is often reactivated when cell-mediated immunity declines due to aging or immunosuppression. SAB, TS ⅡA, and CTS in this study ameliorate the increasing SA-β-gal positive cells which is ascribed to the cellular senescence induced by HCMV infection (Fig. 5A and B). Meanwhile, they also alleviate the degree of DNA damage reflected by SAHF (Fig, 5 C). The results above demonstrate that SAB, TS ⅡA, and CTS could alleviate the degree of cellular senescence in HCMV-infected cells owing to the antiviral properties. Although S. miltiorrhiza has anti-inflammatory and antioxidant properties [21], whether SAB, TS ⅡA, and CTS can alleviate the inflammation caused by HCMV infection, alleviate the ‘cytokine storm’ and thereby facilitate the recovery after the infection is a meaningful topic that deserves further exploration in the next step.

There are two major pathways, p53/p21 and p16/pRb, which play a role in the occurrence and development of cellular senescence [50]. As for HCMV, IE1 promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. This event results in the stimulation of p53 activity, leading to p53/p21-dependent inhibition of cell cycle progression [69], meanwhile, IE1 interacts with p53 and inhibits p53-dependent transactivation by a mechanism different from that of IE2 [70]. IE2 blocks cell cycle progression at the beginning of S phase, which results in cell cycle arrest through p53 signal pathway [71, 72]. Finally, the interaction of IE1, IE2 and p53 lastly initiates the premature cellular senescence in HCMV-infected cells. In addition, HCMV triggers upregulation of p16 expression during the initial stage of infection which in turn plays a positive role for HCMV replication. Therefore, HCMV may exploit the p16-pRb axis to stimulate the senescence program favorable for its replication [47]. In our study, we observe the upregulation of p16, p21, and p53 in HCMV infected cells, but the expression level of these direct markers of cellular senescence reduced dramatically when treated with SAB, TS ⅡA, and CTS. These results above further validated the inhibitory effects of SAB, TS ⅡA, and CTS against HCMV replication from other perspectives. Meanwhile, decreased p16 expression caused by SAB, TS ⅡA, and CTS treatment in HCMV-infected cells in this study would partly in turn affect the subsequent proliferation of the virus.

Oxidative stress, either oxidizing products of the cell metabolism (e.g., ROS) or known oxidative agents (e.g., H₂O₂), has always been one of the major theories of cellular senescence [60, 73]. During the progress of HCMV infection, ROS enhances HCMV infection through the activation of p38-MAPK signaling pathway [74]. Decreased ROS production caused by SAB, TS ⅡA, and CTS treatment in HCMV-infected cells would partly in turn affect the subsequent proliferation of the virus and might be responsible for the antiviral effect.

This study also has limitations. We conducted all the experiments using the same type of cells (Human diploid fibroblasts, WI-38 cells), and did not extend to different cell types which HCMV was known to infect (epithelial cell, dendritic cell, monocytes and endothelial cells). Secondly, due to the requirements of laboratory biosafety and the availability of experimental resources, all the experiments were conducted using HCMV lab strain (Towne), and did not involve other types of lab strains, clinical strains, or clinical variant strains. Meanwhlie, the direct antiviral mechanism of SAB, TS ⅡA, and CTS was worth carrying out gradually in the future, especially the relationship among the SAB, TS ⅡA, CTS and cellular senescence.

Conclusions

Our study demonstrated that SAB, TS ⅡA, and CTS, compounds from an important TCM, S. miltiorrhiza, exhibited potent suppressive effects on the HCMV infection in WI-38 cells for the first time. Subsequent further studies had shown that they mainly play an antiviral role at Post-Entry stage. The antiviral effect of these compounds could alleviate the degree of cellular senescence in HCMV-infected cells. This study facilitates a broad understanding of the anti-HCMV compounds of S. miltiorrhiza, and sheds light on the further development in the therapy against HCMV.

Abbreviations

HSV-1

Herpes simplex virus 1

qPCR

Quantitative polymerase reaction

FBS

Fetal bovine serum

HCMV

Human cytomegalovirus

SAAS

Salvianic acid A sodium

TS I

Tanshinone I

SAB

Salvianolic acid B

TS ⅡA

Tanshinone ⅡA

CTS

Cryptotanshinone

DMSO

Dimethyl sulfoxide

SDS-PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SAHF

Senescence-associated heterochromatin foci

ROS

Reactive oxygen species

dsDNA

Double-stranded DNA

GCV

Ganciclovir

VAV

Valganciclovir

FOS

Foscarnet

CDV

Cidofovir

LEV

Letermovir

MAV

Maribavir

TCM

Traditional Chinese medicine

PD

Population doubling

PVDF

Polyvinylidene fluoride

DMEM

Dulbecco’s Modified Eagle’s Medium

SA-β-gal

Senescence-associated β-galactosidase

ROS

Reactive oxygen species

MOI

Multiplicity of infection

SD

Standart deviation

ECL

Enhanced chemiluminescence

PFA

Paraformaldehyde

TCID₅₀

Tissue culture infective dose

IFA

Indirect immunofluorescence assay

H₂DCFDA

2',7'-dichlorodihydrofluorescein diacetate

CPE

Cytopathic effects

CC₅₀

Half cytotoxic concentration

BF

Bright field

VIS

Virus-induced senescence

Author contributions

Sanying Wang: Conceptualization, Methodology, Investigation, Formal analysis, Validation, Funding acquisition, writing - review and editing; Xuqiang Zhou: Methodology, Investigation, Formal analysis, Writing - Original Draft; Zheng Lin: Formal analysis, Validation; Huili Su: Methodology, Investigation; Xinna Wu: Formal analysis, Investigation; Yunchuang Chang: Methodology, Investigation; Yicheng Fu: Methodology, Investigation; Tianjun Zhu: Methodology, Investigation; Chuan Sun: Investigation, Funding acquisition; Jing Zhang: Investigation; Liqin Li: Validation, Supervision; Ping Lin: Validation, Supervision; Genxiang Mao: Conceptualization, Funding acquisition, Supervision, Writing - Review and Editing.

Funding

This work was supported by Joint TCM Science & Technology Projects of National Demonstration Zones for Comprehensive TCM Reform (GZY-KJS-ZJ-2025-074 and GZY-ZJ-KJ-24055), Zhejiang Provincial Natural Science Foundation of China (LQ20H250002), TCM Key Laboratory Cultivation Base of Zhejiang Province for the Development and Clinical Transformation of Immunomodulatory Drugs (TCM-202402), and the Health Bureau of Zhejiang Province (2022KY009 and 2023KY432).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors approved the manuscript for publication.

Competing interests

The authors declare no competing interests.