Decoction of heat-clearing, detoxifying and blood stasis removing relieves acute soft tissue injury via modulating miR-26b-5p/COX2 axis to inhibit inflammation

Acute soft tissue injury (STI) is a common exercise injury in clinic, imposing a large burden on society due to the direct healthcare costs and time lost from productive employment [1,2]. Acute STI is characterized by aseptic inflammation and causes injuries in several manners, such as local edema, bruising, pain, muscle fiber fracture and petechiae [3,4]. Inflammation and pain reach at peak following the first 2 days of the injury and then decline quickly [5,6]. Noticeably, targeting the excessive inflammatory cascade has been considered as an effective method to improve acute STI [7,8].

Cyclo-oxygenase 2 (COX2), also called as prostaglandin-endoperoxide synthase 2 (PTGS2), serves as a crucial inflammation factor as same as tumor necrosis factor-α (TNF-α), interleukin (IL)-6 (IL-6) and IL-1β [9,10]. Increased evidence has shown that COX2 inhibitors (coxibs) display a good curative effect on the improvement of acute STI [2,11,12]. Prostaglandin E2 (PGE2), an arachidonic acid metabolite, serves as an inflammatory mediator in STI [8,13,14]. Through binding to one of the four downstream receptors (EP1–EP4), which belong to the superfamily of G protein-coupled receptors, PGE2 is closely involved in the inflammatory responses [15,16]. Targeting COX2 and PGE2 might be meaningful in inhibition of the excessive inflammation and the following alleviation of acute STI.

Traditional Chinese medicine (TCM) refers to the herbs and formulae which are made based on the Five Elements theory, including Metal, Wood, Water, Fire and Earth. Up to now, TCM has been applied for few thousand years in China and other Southeast Asian countries to prevent or cure almost all types of diseases, including the inflammation-related diseases [17–19]. Huanglian-Jie-Du-Tang, a decoction made up of heat-clearing and detoxifying TCM-relevant herbs such as Coptis chinensis, Cortex Phellodendri, Scutellaria baicalensis and Fructus gardenia has been identified to inhibit inflammation in inflamed rats, accompanied with decreased levels of macrophage inflammatory protein-2 (MIP-2), TNF-α, IL-6, IL-1β and IL-10 [20]. As acute STI is characterized with excessive inflammatory responses, we conjecture the herbs made up of Huanglian-Jie-Du-Tang which have a good effect on mitigating acute STI. Here, we explored the effect of heat-clearing, detoxifying and blood stasis removing decoction (HDBD), to which was added Astragalus membranaceus, Garden burnet, Rheum officinale, Carthamus tinctorius L. and Borneol to Huanglian-Jie-Du-Tang recipe on acute STI.

HDBD contained 30 g Coptis chinensis, 10 g Cortex Phellodendri, 10 g Scutellaria baicalensis, 10 g Fructus gardenia, 20 g Astragalus membranaceus, 10 g Garden burnet, 10 g Rheum officinale, 5 g Carthamus tinctorius L. and 3 g Borneol which were added to 500 ml water and boiled until the volume was 100 ml.

Thirty male Sprague–Dawley (SD) rats (250–300 g) aged 6–7 weeks were provided by Nanjing Junke Biological Co., LTD. (Jiangsu Province, China) for use in the present study. All animals were maintained in a temperature-controlled condition (24 ± 2°C), with free access to water and chow in a 12-h/12-h light/dark cycle.

The human dermal microvascular endothelium cell line HMEC-1 was purchased from American Type Culture Collection (ATCC, Manassas, VA, U.S.A.). The rat vascular endothelium cell line RAOEC was purchased from Shanghai Jingkang Biological Co., LTD (Shanghai, China). HMEC-1 and RAOEC cells were cultured in MCDB131 (without l-Glutamine), with 10 ng/ml Epidermal Growth Factor (EGF), 1 µg/ml Hydrocortisone, 10 mM Glutamine and 10% (v/v) fetal bovine serum (FBS), all obtained from ATCC.

For HDBD intervention, the cells were incubated with 3:1 (v/v) culture medium/HDMD for 24 h.

The miR-26b-5p mimic/inhibitor (mimic/inhibitor-miR-26b-5p), COX2 overexpressing plasmid (OE-COX2) and the negative control vectors (mimic/inhibitor-NC and OE-NC) were designed and synthesized by GenePharma Co., LTD. (Shanghai, China). Lipofectamine™ 2000 transfection reagent (Invitrogen, Waltham, MA, U.S.A.) was applied for cell transfection based on the instruction book.

Following 1 week of acclimatization, the SD rats were divided randomly into three groups: sham group (saline 0.5 ml), model group (saline 0.5 ml) and HDBD group (HDBD 0.5 ml), with 10 rats in each group. One percent pentobarbital was applied to anesthetize rats intraperitoneally. The hair removal of medial thigh of the knee joint was carried out by using the depilatory cream. Then, the STI models were established with the help of a self-made hammer according to a previous study [3]. A hammer weighing ∼270 g with a bottom-surface radius of 0.6 cm, was dropped from a height of 30 cm to beat the medial thigh (1 cm from the knee joint) on the right side. Rats in the sham group were hit by a marker pen from same height. No bone fracture was caused. Following 30 min of the strike, rats in the sham and model groups were embrocated 0.5 ml saline on the injury area, while HDBD group was embrocated 0.5 ml HDBD once a day for a total of 5 days. At 1, 3, 6 and 10 days after the strike, the temperatures of the healthy side and the affected side were measured using an infrared thermometer. Then, the rats were killed by exsanguination, and the injured muscle tissues were collected and washed with ice-cold saline, and submitted for testing.

The muscle tissues were fixed in 4% paraformaldehyde, and embedded in paraffin. The 5-μm sections were cut and stained with Hematoxylin–Eosin (HE). Slides were scanned and images were taken under an optical microscope (200×; Olympus Corporation, Tokyo, Japan).

Following being homogenized with PBS, the levels of TNF-α, IL-6, IL-1 and PGE2 in the muscle tissues were measured by using enzyme-linked immunosorbent assay (ELISA) Kits (Abcam, Cambridge, MA, U.S.A.), as well as in cell supernatants in the light of the manufacturer’s protocols.

Total RNA was extracted from muscle tissues and cells with TRIzol reagent (Life Technologies, Waltham, MA, U.S.A.) referring to the manufacturer’s manual. Then, the cDNA was produced using a PrimeScript™ First Strand cDNA Synthesis Kit (Takara, Dalian, China). After that, the cDNA was served as substrate for quantitative polymerase chain reaction (qPCR) with SYBR Premix ExTaq (Takara) on an ABI 7900 system (Applied Biosystems, Foster City, CA, U.S.A.). 2^−ΔΔC_t method was used to calculate the relative levels of mRNAs after being normalized to that of the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

• Homo-COX2-sense-5′-AGTCCCTGAGCATCTACGGT-3′,

• COX2-antisense-5′- AAAGGTGTCAGGCAGAAGGG-3′;

• Rattus-COX2-sense-5′- AAAGGTGTCAGGCAGAAGGG-3′

• COX2-antisense-5′- GGGTGGGCTTCAGCAGTAAT-3′;

• Homo-GAPDH-sense-5′- CACTAGGCGCTCACTGTTC-3′,

• GAPDH-antisense-5′- GAGGGATCTCGCTCCTGGAA-3′;

• Rattus-GAPDH-sense-5′- AGTGCCAGCCTCGTCTCATA-3′,

• GAPDH-antisense-5′- GATGGTGATGGGTTTCCCGT-3′.

Protein samples isolated from tissues and cells were separated by the sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and were transferred on to the PVDF membranes (Millipore, Billerica, MA, U.S.A.). After that, the membranes were incubated with the indicated primary antibodies, including anti-COX2 (cat. no. ab15191) and anti-GAPDH (cat. no. ab181602, 1:5000) antibodies, all purchased from Abcam at 4°C overnight. Then, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated indicated secondary antibodies (1:5000) for 1 h at room temperature. Then, the protein expression signals were measured with the help of an ECL kit (Millipore). ImageJ software (version 1.48; National Institutes of Health) was applied for protein quantification.

Luciferase reporter plasmids including the 3′-UTR of COX2 (called as COX2-WT) and the empty luciferase vector were purchased from the Shanghai GenePharma Co., Ltd (Shanghai, China). Mutations were made within the predicted target sites between miR-26b-5p and COX2, which was named as COX2-MUT. To start the luciferase gene reporter assay, cells were seeded in 96-well plates and co-transfected with the COX2-WT/COX2-MUT and mimic-NC/mimic-miR-26b-5p with the help of FuGENE FuGENE® HD transfection reagent (Promega, Madison, WI, U.S.A.). After 48 h of the above treatment, the luciferase activity in each well was measured by using Luciferase Assay Kit (Promega).

All data are expressed as mean ± SD. Statistical analyses were performed using one-way analysis of variance and Student’s t tests with the help of GraphPad Prism 5.0 software. Value of P<0.05 was considered significant difference.

Compared with the model group, the temperature difference between the healthy side and affected side was reduced when the STI mice were given HDBD treatment (Figure 1A). In addition, HDBD treatment significantly reduced the levels of inflammatory factors, including TNF-α, IL-6, PGE2 and IL-1β in the muscle tissues caused by hammer beating (Figure 1B–E). Moreover, tissue edema and the inflammatory cell infiltration were observed in the model group, which were mitigated following HDBD intervention, as shown in Figure 1F. These results demonstrated that HDBD could assuage acute STI and inhibit the excessive inflammatory responses.

To uncover whether miR-26b-5p/COX2 axis is involved in HDBD-mediated STI alleviation, we detected the effects of HDBD intervention on the expression of miR-26b-5p and COX2 in the muscle tissues from the affected thigh. Compared with the sham group, miR-26b-5p level was reduced in the model group while COX2 expression was increased, whereas HDBD intervention reversed these tendencies (Figure 2A,B). This result suggested that miR-26b-5p and COX2 might be involved in HDBD role in assuaging acute STI.

Next, we explored the relationship between miR-26b-5p and COX2 in vitro. Figure 3A showed the putative binding sites between miR-26b-5p and the 3′UTR of COX2 in human and rats. Compared with the mimic-NC group, transfection with mimic-miR-26b-5p induced an obvious increase in miR-26b-5p level in RAOEC and HMEC-1 cells (Figure 3B,C). The luciferase gene reporter assay showed that up-regulation of miR-26b-5p caused a dramatic reduction in the luciferase activity, whereas it was abolished when the binding sites of miR-26b-5p in the 3′UTR of COX2 were mutated (Figure 3D,E). In addition, miR-26b-5p overexpression apparently decreased COX2 expression at both mRNA and protein levels in RAOEC and HMEC-1 cells (Figure 3F–I). These findings confirmed that COX2 was a target of miR-26b-5p in human and rat species.

We then investigated the role of miR-26b-5p/COX2 axis in the inflammatory reactions in vitro. COX2 expression was significantly increased following cell transfection with OE-COX2 in RAOEC and HMEC-1 cells (Figure 4A,B), whereas this effect was abolished after miR-26b-5p overexpression (Figure 4C–F). In addition, miR-26b-5p overexpression reduced the levels of PGE2, TNF-α, IL-6 and IL-1β in the supernatants of RAOEC and HMEC-1 cells, whereas COX2 up-regulation weakened this role (Figure 4G–N). These results demonstrated that miR-26b-5p repressed inflammatory responses via targeting COX2.

Next, we explored whether miR-26b-5p/COX2 axis was involved in HDBD-mediated STI improvement in vitro. HDBD intervention increased miR-26b-5p expression and decreased COX2 expression in RAOEC and HMEC-1 cells, whereas inhibitor-miR-26b-5p reversed this effect (Figure 5A–D). Accordingly, the reduced levels of PGE2, TNF-α, IL-6 and IL-1β induced by HDBD treatment were reversed when miR-26b-5p was down-regulated (Figure 5E–L). These results suggested that HDBD intervention inhibited inflammation via modulating miR-26b-5p/COX2 axis.

The early phase of STI is featured with excessive production of pro-inflammatory factors and successive secondary damage [21]. Although inflammation is necessary to the healing process and benefits wound healing [22], it is indeed harmful when inflammatory responses get out of control during acute STI [23]. In the current study, we explored the function and mechanism underlying HDBD in the treatment of acute STI. The in vivo assay showed that HDBD intervention significantly improved the acute STI through inhibiting the excessive inflammatory responses. The in vitro assay demonstrated that HDBD served as a regulator of miR-26b-5p/COX2 axis to inhibit inflammatory responses.

Evidence has demonstrated that several kinds of TCM exert good effects on alleviation of acute STI. Hydroxysafflor yellow A, the effective flavonoid of dried safflower, attenuated pathologic changes in strike-induced soft tissue inflammation [3]. Wang et al. [23] found that Xiangqing Anodyne Spray suppressed STI-mediated muscle swelling, pro-inflammatory mediator productions, oxidative stress and severe pathological changes in the injured muscle tissue. Huanglian-Jie-Du-Tang, a heat-clearing and detoxifying decoction exerts an inhibitory role in inflammatory responses in different kinds of diseases, such as arthritis [24], liver injuries [25] and polymicrobial sepsis [26]. On the basis of Huanglian-Jie-Du-Tang, we added Astragalus membranaceus, Garden burnet, Rheum officinale, Carthamus tinctorius L. and Borneol to form HDBD. Due to the effects of removing blood stasis, stopping blood and/or protecting brain from injury of Fructus gardenia, Garden burnet, Rheum officinale, Carthamus tinctorius L. and Borneol [27–30], we speculated that HDBD might play a role in anti-inflammation and removing blood stasis. As expected, our results showed that HDBD intervention significantly assuaged the tissue edema and inflammatory cell infiltration in the acute STI model of rats induced by strike, accompanied by the reduced levels of pro-inflammatory factors, such as TNF-α, IL-6, IL-1β and PGE2.

COX is an enzyme which converts arachidonic acid into PGs, has been found to have two isoforms, COX1 and COX2. COX2 is responsible for production of large amounts of pro-inflammatory PGs at the inflammatory site, especially PGE-2, which can work synergistically with other inflammatory factors to exacerbate inflammatory responses [31,32]. In the present study, our results demonstrated that COX2 level was significantly increased in rat injured tissues induced by strike, and was rescued following HDBD administration, indicating that COX2 might be involved in HDBD-mediated mitigation of acute STI. Further exploration showed that COX2 was a target of miR-26b-5p in both human and rat cells. MiR-26b-5p level was demonstrated to be increased in subcutaneous adipose tissue underneath lesional psoriasis skin [33]. Moreover, it has been reported that miR-26b-5p binds to the neutral cholesterol ester hydrolase 1, an enzyme essential for cholesterol efflux which is of importance in the pathogenesis of atherosclerosis as its defection induces excess cholesterol accumulation and foam cells formation, linking miR-26b-5p to inflammation [33–35]. In the current study, we observed that miR-26b-5p level was significantly decreased in rat injured tissues, and was rescued following HDBD administration, which showed opposite to the expression level of COX2. Moreover, we found that miR-26b-5p decreased COX2 expression to repress the production of PGE2, TNF-α, IL-1β and IL-6. However, COX2 overexpression impaired the anti-inflammatory effects of miR-26b-5p in vitro, implying that miR-26b-5p inhibited inflammation by targeting COX2.

Additionally, we investigated whether miR-26b-5p/COX2 axis was involved in HDBD intervention-mediated inflammation repression. The results showed that inhibition of miR-26b-5p dramatically reversed the reductions in the production of PGE2, TNF-α, IL-1β and IL-6 in RAOEC and HMEC-1 cells induced by HDBD treatment. These results illustrated that HDBD intervention inhibited inflammation via regulating miR-26b-5p/COX2 axis.

In conclusion, the present study uncovered that HDBD could relieve acute STI via modulating miR-26b-5p/COX2 axis to inhibit inflammatory responses. The current study shows that HDBD may be a new effective drug for the treatment of acute STI through inhibiting the excessive inflammation. Collectively, our findings demonstrate the theoretical basis for the clinical use of HDBD in treating acute STI, and improve our understanding of the occurrence and development in acute SCI.

All supporting data are included within the main article and its supplementary files.

The authors declare that there are no competing interests associated with the manuscript.

This work was supported by the Scientific Research Item of Traditional Chinese Medicine Bereau of Guangdong Province [grant number 20181228].

Shunwan Jiang provided the idea and did most of the experiments. Zhi Chen, Wenqiang Lai and Qingchun Mai collected and analyzed the data. Dayu Chen and Shufen Sun analyzed the data. Yong Zhang wrote the manuscript.

The animal experiment was performed in the Fourth Clinical Medical College of Guangzhou University of Chinese Medicine and Shanghai Genmed Biological Technology Co., LTD (Shanghai, China). All procedures involving animals obtained the approval of the Ethics Committee of the Fourth Clinical Medical College of Guangzhou University of Chinese Medicine (number [2018]3).

ATCC

American Type Culture Collection

COX2

cyclo-oxygenase 2

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

HDBD

heat-clearing, detoxifying and blood stasis removing decoction

IL

interleukin

PGE2

prostaglandin E2

SD

Sprague–Dawley

STI

soft tissue injury

TCM

traditional Chinese medicine