The Characteristics and Functions of Orally Absorbed Herbal Decoction-Borne Plant MicroRNAs

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Ethics approval and consent to participate
• Qingfei Paidu and Qingre Huashi Kangdu decoctions
• Virus
• Oral absorption and kinetics of herbal decoction-borne plant miRNAs in humans
• Oral absorption of herbal decoction-borne plant miRNAs in rats
• Illumina deep sequencing
• Logistical repression analysis to identify the characteristics of the orally absorbed decoction-borne plant microRNAs
• Identification of known and novel miRNAs
• Reverse transcription-quantitative polymerase chain reaction of MIR-6240 – 3 p
• Antiviral effect of MIR-6240 – 3 p on HCoV-229E in vitro
• Reverse transcription-quantitative polymerase chain reaction of HCoV-229E RNA
• Statistical analysis
• Availability of data and materials
• Contributorsʼ Statement
• References

Abstract

Herbal decoctions always contain numerous plant microRNAs, and some of these can be absorbed orally to exert cross-kingdom gene regulation. However, little is known about which specific types of herbal decoction-borne plant microRNAs are more likely to be absorbed. Thus, two antiviral herbal decoctions, Qingfei Paidu and Qingre Huashi Kangdu, were administered to human volunteers and rats, respectively, to investigate the characteristics of orally absorbed decoction-borne plant microRNAs. MIR-6240 – 3 p was identified as an absorbed plant microRNA in humans and is most highly expressed in Qingfei Paidu decoction. Therefore, the kinetics of MIR-6240 – 3 p were monitored in humans following the administration of the Qingfei Paidu decoction, and its antiviral effect on human coronavirus type 229E (HCoV-229E) was examined in vitro. There were 586 176 small RNAs identified in Qingfei Paidu decoction, of which 100 276 were orally absorbed by humans. In the Qingre Huashi Kangdu decoction, 124 026 small RNAs were detected, with 7484 being orally absorbed by rats. Logistical repression analysis revealed that absorbable plant small RNAs in both humans and rats presented higher expression levels, greater minimum free energy, and increased AU/UA frequencies compared to nonabsorbable plant small RNAs. The amount of MIR-6240 – 3 p in humans increased between 1 and 3 h after the administration of the Qingfei Paidu decoction. In addition, MIR-6240 – 3 p significantly reduced the RNA copy number and TCID₅₀ of HCoV-229E in vitro. These results suggest that herbal decoction-borne plant small RNAs with a higher expression level, greater minimum free energy, or an increased AU/UA frequency are more likely to be orally absorbed and could potentially mediate cross-kingdom gene regulation.

Introduction

Cross-kingdom gene regulation by plant microRNAs (miRNAs) was first introduced in 2012, when Zhang and his team discovered that MIR-168a in dietary rice could cross the inter-kingdom boundary and function in rat liver by targeting the low-density lipoprotein receptor adapter protein 1 message RNA (mRNA) [1]. Since then, the possibility of cross-kingdom gene regulation via orally acquired plant miRNAs has been extensively studied and debated. Research has shown that plant miRNAs could enter the animal body through multiple routes, including gastrointestinal tract (GT), respiratory tract, venous injection, and so on [2]. Among them, dietary plant miRNAs absorbed through the GT have garnered the most attention due to concerns over the safety of genetically modified plant foods [3]. Moreover, orally absorbed plant miRNAs recognize targets based on principles of miRNA-target recognition in animals [2], which is different from those in plants [4], [5].

Current research suggests that the cross-kingdom gene regulation can occur for certain plant miRNAs (though not all) [2]. However, little is known about which specific types of plant miRNAs are most likely to be absorbed and exert cross-kingdom effects [2]. Plant miRNAs must withstand the rigorous physical and physiological conditions to gain entry into the bodyʼs circulatory system, such as exposure to high temperatures and pressure during cooking or decoction, digestion by gastric acid, and degradation by nucleases in the GT [6], [7], [8], [9], [10]. Following these challenges, plant miRNAs can be internalized through the SID-1 transmembrane family member 1 (SIDT1)-mediated transport pathway, which is located in the plasma membrane of gastric epithelial mucous cells [11]. The absorption rate of plant miRNAs within the GT is relatively low, ranging from 0.04 to 4.5% [6], [7], [8]. As a result, the expression level of plant miRNAs in the plant sample is a critical factor to ensure that a sufficient amount of these molecules is going to be absorbed for potential cross-kingdom regulatory effects. Additionally, the inherent stability of plant miRNAs is another key factor in enhancing their resilience to those rigorous conditions, including the methylation of the 2′-OH group of the last nucleotide ribose of miRNAs [12], [13], the minimum free energy required for forming secondary structures [14], [15], [16], the frequency of AU/UA dinucleotides [15], [17], or the percentage of a specific single nucleotide within the miRNAs [18]. Hence, the goal of the present study was to investigate the effects of these variables on the oral bioavailability of plant miRNAs.

Herbal decoctions often contain a significant number of plant miRNAs, some of which can be absorbed orally in the animal body and participate in cross-kingdom gene regulation [19], [20], [21], [22]. These absorbable and nonabsorbable plant small RNAs (sRNAs, including miRNAs) can provide valuable data for analysis. In other words, herbal decoctions may serve as an ideal study material to address our research question. Qingfei Paidu (QFPD) decoction and Qingre Huashi Kangdu (QRHS) decoction have been widely used in China for treating coronavirus disease 2019 (COVID-19) [23], [24], [25]. Taking these two decoctions as the materials, we analyzed the characteristics of orally acquired herbal decoction-borne plant sRNAs in vivo, as well as the cross-kingdom gene regulation of MIR-6240 – 3 p in vitro – one of the absorbed and most highly expressed miRNAs found in QFPD decoction.

Results

To probe into the characteristics of orally absorbed herbal decoction-borne plant miRNAs in humans, we recruited three healthy volunteers, aged 24 – 25 years, and administered 180 mL QFPD decoction orally. A total of 586 176 sRNAs were detected in the QFPD decoction (n = 2). Of these, 93 131 sRNAs were detected in serum samples taken at 0.5 and 1 h post-administration, which were absent in preadministration serum samples. There were 7145 sRNAs found in all serum samples, with their levels increasing 0.5 or 1 h post-administration. Thus, a total of 100 276 sRNAs were classified as absorbed QFPD-borne plant sRNAs. Furthermore, 416 961 sRNAs were only detected in the QFPD decoction and deemed nonabsorbed.

A t-test indicated that three factors were statistically higher in the absorbed plant sRNAs than those in the nonabsorbed plant sRNAs, including the expression levels of sRNAs in the decoction (expression level), the minimum free energy of sRNAs required for forming a secondary structure (minimum free energy), and the frequency of AU/UA dinucleotides in the sRNAs (AU/UA frequency). These results suggested a potential correlation between these three factors and the oral absorption of decoction-borne plant sRNAs. Consequently, we further analyzed these factors by using a logistic regression model.

The logistical regression analysis revealed that the relationship between the likelihood of plant sRNA absorption and the three examined factors can be expressed using the following equation: Odds of absorption = − 1.544 + 0.14 × Expression level + 0.05 × Minimum free energy + 0.03 × AU/UA frequency ([Table 1]). Each factor positively influenced the absorption of plant sRNAs; specifically, the higher the expression level, the greater the minimum free energy or increased AU/UA frequency corresponded to a higher likelihood of oral absorption by humans ([Table 1]). For instance, if the expression level of an observed sRNA increased by one count per million (CPM), while the other two factors remained constant, the odds of absorption would increase by 1.15 times compared to before. In this equation, the expression levels ranged from 0.06 to 490.13 CPM, the minimum free energy ranged from − 15.06 to 0 kcal/mol, and AU/UA frequencies ranged from 0 to 18.33%. No significant correlation was noted between the frequency of any nucleotide (i.e., A, U, C, or G) in the sRNAs and oral absorption of plant sRNAs in humans.

Consistent results were observed in rats administered the QRHS decoction. Specifically, a total of 124 026 sRNAs were detected in the decoction (n = 2). Among them, 7133 sRNAs were identified in serum samples from the decoction group at 3 or 6 h post-administration, which were absent in the control group serum samples. There were 351 sRNAs detected in all serum samples, which showed increased levels in the decoction group compared to the control group. Therefore, these two types of sRNAs were classified as absorbed plant sRNAs. In addition, the remaining 100 274 sRNAs were only detected in the QFPD decoction and deemed nonabsorbed.

As mentioned above, the characteristics between the two datasets were compared using a t-test and a logistical regression analysis. Briefly, the t-test indicated that the three examined factors were statistically higher among absorbed plant sRNAs than nonabsorbed ones. The logistic regression analysis revealed a relationship between absorption odds of plant sRNAs and the three factors analyzed, expressed as: Odds of absorption = − 2.46 + 0.03 × Expression level + 0.14 × Minimum free energy + 0.07 × AU/UA frequency ([Table 2]). In this equation, the expression levels ranged from 0.09 to 274.59 CPM, the minimum free energy ranged from − 13.83 to 0 kcal/mol, and AU/UA frequencies ranged from 0 to 15.00%. Each factor positively influenced the absorption of plant sRNAs. No significant correlation was noted between the frequency of any nucleotide (i.e., A, U, C, or G) in the sRNAs and oral absorption of plant sRNAs in rats.

MIR-6240 – 3 p was the most highly expressed miRNA in the QFPD decoction, with a sequence of 5′-UUUCUGCCCAGUGCUCUG-3′. The kinetics of MIR-6240 – 3 p were further studied in humans to validate the oral absorption of plant sRNAs. Six healthy volunteers, aged 24 – 25, were recruited and randomly assigned to the treatment group or control group. The treatment group was administered 180 mL of QFPD decoction per person orally, while the control group received 180 mL of warm boiled water per person. Peripheral blood samples were collected from each volunteer at 0, 1, 2, 3, and 4 h after administration.

The levels of MIR-6240 – 3 p in peripheral blood serum were quantified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The standard curve between concentration (pM) and Cycle Threshold (C_T) values obtained by RT-qPCR was expressed as: log₁₀ ^{Concentrations(pM)} = 8.52 – 0.25 × C_T values, R² = 0.99. For the C_T value, the linear range of the standard curve was from 9.66 to 33.25. The C_T values detected in all the samples were from 20.05 to 32.95, falling within the linear range. The C_T values of the negative control were greater than 40 (i.e., undetermined), distinguishing them from the serum or QFPD decoction samples. These indicated that the RT-qPCR system was specific and sensitive for MIR-6240 – 3 p detection. Based on the standard curve, the concentration of MIR-6240 – 3 p in the QFPD decoction ranged from 1820.20 to 3844.76 pM, while the concentration in human serum ranged from 1.71 to 10.26 pM.

It was observed that in the treatment group, the level of MIR-6240 – 3 p, relative to snRNA U6, was significantly elevated at 1 h after the administration of 180 mL QFPD decoction compared to baseline (the relative level of MIR-6240 – 3 p before administration). This elevation persisted until 3 h post-administration, after which it sharply fell at 4 h. In the control group, the level of MIR-6240 – 3 p remained unchanged from 0 to 3 h following the parallel administration of 180 mL of warm boiled water but significantly dropped at 4 h. These results indicated that the elevated levels of MIR-6240 – 3 p in the treatment group at 1 and 3 h post-administration were attributed to the QFPD decoction taken orally, while the decrease at 4 h in both groups was likely due to metabolic processes in the body ([Fig. 1]).

These results, presented in [Tables 1] and [2] and [Fig. 1], demonstrate that herbal decoction-borne plant sRNAs could be orally absorbed in animals. Plant miRNAs higher expression level, greater minimum free energy, or increased AU/UA frequency were more likely to be absorbed, as indicated in [Fig. 2].

Given that the QFPD decoction was recommended for COVID-19 treatment and MIR-6240 – 3 p was the most highly expressed plant miRNA in this decoction, we synthesized MIR-6240 – 3 p mimics to disrupt the replication of HCoV-229E in human embryo lung cells (MRC-5). In the infection group, the average viral titer was 25 279.28 50% Tissue Culture Infectious Dose (TCID₅₀)/mL but significantly decreased to 3020.98 TCID₅₀/mL with MIR-6240 – 3 p mimics (p < 0.05), as depicted in the MIR-6240 – 3 p group ([Fig. 3 a]). This represented a reduction of 89.05% (calculated as 1 – 3020.98/25 279.28 × 100%). The average viral titer in the MIR-NC group showed no statistical difference from the infection group (p > 0.05), indicating that the observed reduction in viral titers was specifically due to MIR-6240 – 3 p rather than a general stress response within the cells. A similar trend was observed in the viral RNA copies ([Fig. 3 b]).

In further experiments, it was found that MIR-6240 – 3 p inhibitors could reverse the antiviral effect of MIR-6240 – 3 p mimics on HCoV-229E. Specifically, the average viral titer decreased from 44.76 million TCID₅₀/mL in the infection group to 7.11 million TCID₅₀/mL (p < 0.05) under the effect of MIR-6240 – 3 p mimics, as shown in the MIR-6240 – 3 p group ([Fig. 4 a]). In contrast, in the MIR-6240 – 3 p inhibitor group, the average viral titer was restored to 44.62 million TCID₅₀/mL by the MIR-6240 – 3 p inhibitor, which was statistically indistinguishable from the levels observed in the infection group (p > 0.05). A similar trend was observed in the viral RNA copies ([Fig. 4 b]). These results suggest that MIR-6240 – 3 p can inhibit HCoV-229E in vitro ([Figs. 3] and [4]).

Discussion

To achieve cross-kingdom gene regulation in animals, exogenous plant miRNAs must overcome at least four physiological barriers [2], [26]: (1) Selective absorption of plant miRNAs in GT. Currently, it has been observed that the absorption of plant miRNAs in GT is selective [7], [27], but which specific types of plant miRNAs are selectively absorbed, and the mechanisms involved remain unknown. (2) Argonaute (AGO) specificity. For miRNAs to function, they must be loaded onto AGO proteins, but the loading process of miRNAs onto AGO proteins is also selective. (3) Complementarity between miRNAs and target mRNAs. Based on the mechanism of miRNA action, a functional exogenous plant miRNA needs sufficient base pairing with the target mRNA to form a stable AGO-miRNA-mRNA complex. (4) Accessibility of target sites in mRNAs. A variety of factors, like secondary structures at target sites, can influence the accessibility of miRNAs to their targets, potentially blocking them from binding.

The present study primarily focused on the first physiological barrier, investigating which types of herbal decoction-borne plant miRNAs can be selectively absorbed in the GT. For the first time, it was discovered that herbal decoction-borne plant sRNAs with a higher expression level, greater minimum free energy, or increased AU/UA frequency were more likely to be absorbed orally ([Tables 1] and [2]). We conducted a systematic review of the characteristics of plant-derived miRNAs that have been reported to be absorbed orally by humans or animals. Twenty-four plant miRNAs were identified and analyzed, including MIR-2911, MIR-159, MIR-172a, MIR-167, MIR-168a, MIR-156a, MIR-166a, MIR-472a, MIR-951, MIR-162a, MIR-390a, MIR-528, MIR-157a, MIR-158a, MIR-159a, MIR-160a, MIR-163a, MIR-169a, MIR-824, MIR-170a, and MIR-395a [1], [6], [7], [28], [29], [30], [31]. The minimum free energy and AU/UA nucleotide frequency for these miRNAs were computed using the methods described in this paper. Interestingly, these plant miRNAs are known to be highly expressed in plants and exhibit higher minimum free energy or an increased AU/UA frequency, which supports our findings (detailed results not presented here).

The correlation between a higher expression level of plant miRNAs and their absorption was straightforward; the higher expression level a plant miRNA had, the more likely it was to be absorbed. This was why researchers often selected highly expressed plant miRNAs to investigate their cross-kingdom gene regulation [1], [21], [28], [32]. However, it was challenging to explain the other two factors. Typically, sRNAs with a greater minimum free energy of their secondary structure are indicative of reduced stability, making them more prone to unfolding into a linear configuration [15]. Based on this, we inferred that plant sRNAs possessing a greater minimum free energy are more likely to be present in a linear state, potentially facilitating their uptake by the GT. This hypothesis is consistent with the earlier findings of Zhang et al., who suggested that the single-stranded mature form of MIR168a is the probable form absorbed by the GT [1].

Regarding the AU/UA frequency, it is well established that the high frequency of AU/UA motifs in mRNAs enables their specific recognition by particular RNA-binding proteins (RBPs), such as human antigen R (HuR), AU-rich element RNA-binding protein 1 (AUF1), KH-type splicing regulatory protein (KSRP), and others. This specific recognition by RBPs is essential for the nuclear export of mRNAs to cytoplasm [33], [34], [35]. Similarly, AU/UA motifs within miRNAs may also play a role in regulating their cellular secretion into the circulatory system [36]. As reported by Lino et al., in 2024, AU/UA-rich motifs within miRNAs can be specifically recognized by RBPs, including heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), kinase-docking RNA-binding protein with SH3 domain 1 (KHDRBS1), KH-type splicing regulatory protein (KHSRP), pumilio RNA-binding protein 1 and 2 (PUM1 and PUM2), and others [36]. The interaction between these RBPs and the AU/UA motifs in miRNAs facilitates the packaging of miRNAs into small extracellular vesicles (sEVs) [36], which are subsequently secreted into the circulatory system [37]. As aforesaid, plant miRNAs can be absorbed by gastric epithelial mucous cells through the mediation of SIDT1 [11]. Within these cells, the absorbed plant miRNAs are released into the bloodstream via sEVs [11]. Combining all this knowledge, it is reasonable to propose that plant miRNAs with a higher AU/UA frequency are more likely to be selectively packaged into sEVs, with the assistance of RBPs, and efficiently secreted (i.e., absorbed) into the bloodstream. This may account for our observation that plant sRNAs with increased AU/UA frequencies were more likely to be absorbed orally. However, further research is needed to validate our hypothesis regarding the sequence-selective absorption mechanism of miRNAs in the human or animal GT. Moreover, apart from facilitating the secretion of miRNAs from gastric epithelial mucous cells to the bloodstream in the form of sEVs, the binding of RBPs to the AU/UA motifs in miRNAs can also prevent the absorbed plant miRNAs from degradation by nucleases in the blood, prolonging their duration of gene regulation.

Additionally, the kinetics of MIR-6240 – 3 p in human serum was mapped ([Fig. 1]), showing a pattern similar to that of lettuce MIR-156a after healthy volunteers consumed lettuce [29]. The kinetics of MIR-6240 – 3 p, a highly expressed plant miRNA in the QFPD decoction, further supported the oral absorption of plant sRNAs. The in vitro antiviral effect of MIR-6240 – 3 p on HCoV-229E was also observed. In the next step, we intended to explore the target of MIR-6240 – 3 p and its antiviral effects on HCoV-229E in vivo.

Collectively, considering the consistent results from both human and rat experiments, it was reasonable to conclude that herbal decoction-borne plant sRNAs with a higher expression level, greater minimum free energy, or increased AU/UA frequency were more likely to be orally absorbed in animals and subsequently mediate cross-kingdom gene regulation.

Materials and Methods

Ethics approval and consent to participate

The in vitro experiment with live human coronaviruses type 229E (HCoV-229E) was carried out in the biosafety level 2 facility at Jiangxi University of Chinese Medicine, which was approved for such use by the Nanchang Municipal Health Commission, Jiangxi Province, China. The human studies were approved by the Medical Ethics Committee of the Affiliated Hospital of Jiangxi University of Chinese Medicine on August 11, 2022 (No. JZFYLL20220727024). All volunteers signed informed consent forms. The rat study was approved by the Ethical Review Committee and Laboratory Animal Welfare Committee at Jiangxi University of Chinese Medicine on November 3, 2022 (No. jzllsc20210054).

Qingfei Paidu and Qingre Huashi Kangdu decoctions

Both QFPD and QRHS decoctions were multiherbal preparations composed of 21 and 10 Chinese medicinal herbs, respectively (Tables 1S and 2S, Supporting Information) [38], [39], and prepared as follows [40]. The QFPD decoction was prepared by first decocting Sheng Shi Gao in 1500 mL of purified water for 5 min, followed by the co-decoction of the remaining 20 raw herbs (Tables 1S, Supporting Information) until the solvent volume reduced to about 200 mL, yielding a concentration of about 1.05 g/mL (211 g/200 mL). For the QRHS decoction, 1200 mL of purified water was added to 10 Chinese medicinal herbs (Tables 2S, Supporting Information), and the mixture was decocted until the solvent volume decreased to around 180 mL, achieving a concentration of about 0.5 g/mL (90 g/180 mL). Both decoctions were stored at 4 °C and used within 1 week. They needed to be warmed before use.

Virus

The HCoV-229E virus was kindly provided by the Chinese Center for Disease Control and Prevention.

Oral absorption and kinetics of herbal decoction-borne plant miRNAs in humans

Two sequential human studies were conducted. In both studies, volunteers had to conform to the following criteria: (1) healthy students aged 18 to 25 years from Jiangxi University of Chinese Medicine, (2) no cold or fever in the past 2 weeks, (3) for female volunteers, not during menstruation, (4) no gastrointestinal disorders, and (5) signed informed consent. Additionally, individuals would be excluded if they exhibited any of the following conditions: (1) fear of needles or the sight of blood or (2) discomfort after administration of the decoction. Eligible volunteers were orally administered either 180 mL of QFPD decoction or warm boiled water. On the day prior to administration, volunteers were instructed to consume a bland diet and ensure adequate sleep. At any stage of the study, participants could withdraw.

The first human study aimed to investigate the absorption of the entire sRNA content in the QFPD decoction. Three volunteers were recruited, each receiving 180 mL of QFPD decoction orally. Blood samples were collected at three time points: 5 min before administration (0 h), and 0.5 and 1 h after administration. Peripheral blood samples were collected from each volunteer. Then, serum samples were isolated from the peripheral blood. The sRNA profiles in both the serum samples and the QFPD decoction were sequenced synchronously. The analysis focused on sRNAs ranging from 16 to 30 nucleotides in length. The following two types of sRNAs were classified as absorbed plant sRNAs: (1) those detected in the QFPD decoction and in serum samples collected at 0.5 or 1 h after administration but absent in serum samples taken at 0 hours and (2) those found in the QFPD decoction and all serum samples, with an increased level in serum samples collected at 0.5 or 1 h post-administration. In contrast, sRNAs that were only detected in the QFPD decoction (i.e., absent in all serum samples) were classified as nonabsorbed plant sRNAs.

The second human study aimed to investigate the kinetics of MIR-6240 – 3 p, the most highly expressed plant miRNA in QFPD decoction, to validate the oral absorption of plant sRNAs observed in the first study. Six volunteers were recruited and randomly divided into a treatment group and a control group. The treatment group was administered 180 mL of QFPD decoction per person orally, while the control group received 180 mL of warm boiled water per person. Peripheral blood samples were collected from each volunteer at the following time points: 5 min before administration (0 h) and 1, 2, 3, and 4 h after administration. The concentration of MIR-6240 – 3 p in the peripheral blood was monitored by means of RT-qPCR.

Oral absorption of herbal decoction-borne plant miRNAs in rats

In order to validate the findings from human studies, we carried out a rat study. Eight female 6-week-old Sprague-Dawley rats, obtained from the National Laboratory Animal Seed Center, were acclimatized for 1 week prior to the experiment under controlled conditions (23 ± 2 °C, 40 – 70% relative humidity, and a 12 : 12 light-dark cycle), with ad libitum access to standard food and water. The rats were then grouped into four pairs based on weight, sex, and cote before being randomly assigned into two groups: the decoction group and the control group. Within each pair, one rat was assigned to the decoction group and the other to the control group. The decoction group received an oral administration of QRHS decoction at a dosage of 2 mL per rat, while the control group was given the same volume of warm boiled water per rat. The rats were housed at the Experimental Animal Science and Technology Center of Jiangxi University of Chinese Medicine. At the time points of 3 and 6 h post-administration, two pairs of rats (four in total) were randomly selected for sampling. The rats were then anesthetized using pentobarbital sodium, and their blood was collected from the abdominal aorta without anticoagulants. Serum was separated from the blood samples by centrifugation at 4 °C at 1600 × g for 10 min. Both the sRNA profiles in the serum samples and the QRHS decoction were sequenced synchronously.

As mentioned above, the sRNAs ranging from 16 to 30 nucleotides in length were analyzed. The following two types of sRNAs were classified as absorbed plant sRNAs: (1) those detected in both the QRHS decoction and the serum samples from the decoction group at 3 or 6 h post-administration but absent in the serum samples of the control group and (2) those found in both the QRHS decoction and serum samples, with an increased level in the serum samples of the decoction group. In contrast, sRNAs that were detected only in the QRHS decoction (i.e., absent in all serum samples) were deemed as nonabsorbed plant sRNAs.

Illumina deep sequencing

Total RNA was extracted using a Total RNA Purification Kit (TRK-1001) following the manufacturerʼs protocol. The quantity and purity of the total RNA were assessed with a Agilent Bioanalyzer 2100 and an Agilent RNA 6000 Nano LabChip Kit. About 1 µg of total RNA was utilized to prepare a small RNA library according to the Illumina TruSeq Small RNA Sample Prep Kit protocol. Subsequently, single-end sequencing (1 × 50 bp) was performed on an Illumina HiSeq 2500 at LC-BIO according to the vendorʼs recommended procedures.

Logistical repression analysis to identify the characteristics of the orally absorbed decoction-borne plant microRNAs

In the logistic regression analysis, four factors were considered: the expression level of plant sRNAs, the minimum free energy of sRNAs, the frequency of AU/UA dinucleotides in sRNAs, and the percentage of specific single nucleotides in the sRNAs.

• Expression level of plant sRNAs: It was normalized as CPM using the formula: read counts of a sRNA/total counts of all detected sRNAs × 1 000 000.

• Minimum free energy of miRNAs: It was estimated using RNAfold software and adjusted to a length of 20 nucleotides using the formula: the estimated minimum free energy of a sRNA/length of the sRNA × 20.

• Frequency of AU/UA dinucleotides: It was calculated as the total counts of AU and UA dinucleotides in either the 5′-3′ or 3′-5′ orientation and then adjusted for a length of 20 nucleotides using the formula: the total counts of AU and UA dinucleotides in a sRNA/length of the sRNA × 20.

• Percentage of specific single nucleotides in sRNAs: It referred to the percentage of total counts of a specific nucleotide (i.e., A, U, C, or G) relative to the length of the sRNA.

  To begin with, a t-test was conducted for each individual factor to identify statistically significant differences between absorbed and nonabsorbed plant sRNAs. The factors that showed significant differences were then selected for logistic regression analysis. In this analysis, these statistically significant factors were treated as independent variables, while the absorbed and nonabsorbed sRNAs were labeled as “1” and “0”, respectively, serving as dependent variables.

Identification of known and novel miRNAs

The known mature miRNAs and precursor miRNAs of the species were retrieved from miRBase established by the University of Manchester. We utilized miRDeep2 to discover novel miRNAs, predict the hairpin structure of precursor miRNAs, and quantify miRNAs. Also, we aligned the herbal sRNAs to the known mature miRNAs and their precursors in miRBase to identify known miRNAs and obtain their counts. Given that miRNAs were evolutionarily conserved, we summarized the numerical counts and read counts of known miRNA families.

Reverse transcription-quantitative polymerase chain reaction of MIR-6240 – 3 p

sRNAs (< 100 nucleotides) from the QFPD decoction were extracted with the Universal Plant MicroRNA Kit (Bioteke Cat # RP5331). Total RNA from serum was extracted with the miRNeasy Mini Kit (Qiagen 217 004). RT-qPCR was performed using the miRNA 1st Strand cDNA Synthesis Kit (Vazyme MR101) for reverse transcription (RT) and the miRNA Universal SYBR qPCR Master Mix (Vazyme MQ101) for quantitative PCR following the manufacturerʼs instructions. In order to calculate the absolute amount of MIR-6240 – 3 p, a series of synthetic MIR-6240 – 3 p oligonucleotides at known concentrations were reverse transcribed and amplified. The absolute amount of MIR-6240 – 3 p was determined in reference to a standard curve. U6 snRNA was used as an internal control. Additionally, reverse transcription was performed using a LongGene A600 Super Gradient Thermal Cycler, and qPCR was performed on a Roche LightCycler 96 Instrument.

The RT-qPCR primer set for MIR-6240 – 3 p included: RT primer: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAGAGC-3′; F-primer for qPCR: 5′-GCGCGTTTCTGCCCAGT-3′; R-primer for qPCR: 5′-AGTGCAGGGTCCGAGGTATT-3′.

The RT-qPCR primer set for snRNA U6 included: RT primer: 5′-AACGCTTCACGAATTTGCGT-3′; F-primer for qPCR: 5′-CTCGCTTCGGCAGCACA-3′; R-primer for qPCR: 5′-AACGCTTCACGAATTTGCGT-3′. The reverse transcription reaction conditions were 25 °C for 5 min, 50 °C for 15 min, and 85 °C for 5 min. The qPCR reaction conditions were one cycle of 95 °C for 1 min, 40 cycles of 95 °C for 10 s and 60 °C for 30 s (fluorescence measured at 60 °C).

Antiviral effect of MIR-6240 – 3 p on HCoV-229E in vitro

MRC-5 cells were employed for the in vitro experiment. To determine the antiviral effect of MIR-6240 – 3 p on HCoV-229E, we conducted two independent cell experiments. The first experiment aimed to evaluate the antiviral effect of MIR-6240 – 3 p mimics on HCoV-229E. The groups included an MIR-6240 – 3 p group, MIR-NC group, infection group, and negative control group. Each group comprised four replicates. The second experiment aimed to investigate whether MIR-6240 – 3 p inhibitors can counteract the antiviral effect of MIR-6240 – 3 p mimics on HCoV-229E. The groups included an MIR-6240 – 3 p inhibitor group, MIR-6240 – 3 p group, infection group, and negative control group. Each group comprised three replicates.

In the first experiment, the MIR-6240 – 3 p group and the MIR-NC group were transfected with MIR-6240 – 3 p mimics and MIR-NC, respectively, using an Invitrogen Lipofectamine 2000, following the manufacturerʼs instructions. In the second experiment, the MIR-6240 – 3 p inhibitor group was sequentially transfected with MIR-6240 – 3 p inhibitor and MIR-6240 – 3 p mimics, while the MIR-6240 – 3 p group received MIR-6240 – 3 p inhibitor NC followed by MIR-6240 – 3 p mimics. All infection and negative control groups in both experiments were treated with DMEM medium. The tested concentration was 13.2 nM.

Then, in each experiment, the transfected groups and the infection groups were exposed to HCoV-229E at a multiplicity of infection (MOI) of 0.1 at 37 °C. The negative control group was synchronously treated with DMEM medium instead of the HCoV-229E solution. After 2 h of infection, the cells were washed with warm PBS three times and then incubated in DMEM medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 2% fetal bovine serum. After 48 h, cell supernatants were collected for RT-qPCR detection of HCoV-229E RNA copies.

The sequences of the synthesized miRNA or sRNAs were as follows: MIR-6240 – 3 p mimics: 5′-UUUCUGCCCAGUGCUCUG-3′; MIR-6240 – 3 p inhibitors: 5′-CAGAGCACUGGGCAGAAA-3′; MIR-6240 – 3 p mimics NC: 5′-UUGUACUACACAAAAGUACUG-3′; MIR-6240 – 3 p inhibitors NC: 5′-CAGUACUUUUGUGUAGUACAA-3′. All sRNAs were modified with 2′-O-methylation.

Reverse transcription-quantitative polymerase chain reaction of HCoV-229E RNA

The copies of the HCoV-229E M gene in the collected supernatants were identified to estimate viral loads. Total RNA was extracted using the SteadyPure Virus DNA/RNA Extraction Kit (Accurate Biotechnology AG21021). RT-qPCR was performed using the HiScript Ⅱ U+ One Step qRT-PCR Probe Kit (Vazyme Q223-01) following the manufacturerʼs instructions. To calculate the absolute copies of the HCoV-229E M gene, a series of RNA standard products (BNCC370733) with known concentrations were reverse transcribed and amplified. The absolute amount of the HCoV-229E M gene was then determined based on a standard curve.

The RT-qPCR primer set for the HCoV-229E M gene included: Probe: 5′-FAM-TTTGACACCTGGGCTAATTGGGA-BHQ1 – 3′; F-primer: 5′-TTGGCCACTCTCGTACTTGCTT-3′; R-primer: 5′-GTTCGAGCACGTCGGAAAAG-3′. The reaction conditions were one cycle of 55 °C for 15 min, one cycle of 95 °C for 30 s, and 40 cycles of 95 °C for 10 s and 60 °C for 30 s (fluorescence measured at 60 °C). RT-qPCR was performed using the Roche LightCycler 96 Instrument.

Statistical analysis

The amount of MIR-6240 – 3 p in serum was determined following normalization to U6 snRNA levels using the 2^−ΔΔCt method. The viral TCID₅₀ and HCoV-229E RNA concentrations were transformed into log2 values. A two-sample t-test was carried out to compare the amounts between groups, with p < 0.05 indicating significant differences.

Availability of data and materials

All related data and materials are included in this paper. All sequencing data have been uploaded to the SRA database (BioProject accession number: PRJNA1147404).

Contributorsʼ Statement

Conception and design: Tielong Xu, Longxue Li, Bin Zheng; data collection: Yating Zhu, Yicheng Yu, Yao Jia, Ziqi Lin, Jinyue Lei, Diyao Wu; analysis and interpretation of data: Tielong Xu, Longxue Li, Bin Zheng, Yating Zhu, Yicheng Yu; statistical analysis: Tielong Xu, Yating Zhu, Yicheng Yu; drafting of the manuscript: Tielong Xu, Longxue Li, Yating Zhu, Yicheng Yu; critical revision of the manuscript: Tielong Xu, Bin Zheng.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Correspondence

Publication History

Received: 08 October 2024

Accepted after revision: 28 January 2025

Accepted Manuscript online:
28 January 2025

Article published online:
26 February 2025

© 2025. Thieme. All rights reserved.

Georg Thieme Verlag KG
Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany

• References

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  CrossrefPubMedSearch in Google Scholar
• 30 Chin AR, Fong MY, Somlo G, Wu J, Swiderski P, Wu X, Wang SE. Cross-kingdom inhibition of breast cancer growth by plant miR159. Cell Res 2016; 26: 217-228
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 37 Wu Q, Li L, Jia Y, Xu T, Zhou X. Advances in studies of circulating microRNAs: Origination, transportation, and distal target regulation. J Cell Commun Signal 2023; 17: 445-455
  CrossrefPubMedSearch in Google Scholar
• 38 Al-Romaima A, Liao Y, Feng J, Qin X, Qin G. Advances in the treatment of novel coronavirus disease (COVID-19) with Western medicine and traditional Chinese medicine: A narrative review. J Thorac Dis 2020; 12: 6054-6069
  CrossrefPubMedSearch in Google Scholar
• 39 Ren W, Ma Y, Wang R, Liang P, Sun Q, Pu Q, Dong L, Mazhar M, Luo G, Yang S. Research advance on Qingfei Paidu decoction in prescription principle, mechanism analysis and clinical application. Front Pharmacol 2020; 11: 589714
  CrossrefPubMedSearch in Google Scholar
• 40 Xin S, Cheng X, Zhu B, Liao X, Yang F, Song L, Shi Y, Guan X, Su R, Wang J, Xing L, Xu X, Jin L, Liu Y, Zhou W, Zhang D, Liang L, Yu Y, Yu R. Clinical retrospective study on the efficacy of Qingfei Paidu decoction combined with Western medicine for COVID-19 treatment. Biomed Pharmacother 2020; 129: 110500
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material