Codonopsis Decoction Inhibits Aspirin-Induced Gastric Mucosal Injury in Rats by Regulating COX, COX-1, COX-2, TNF-α, IL-6 and TGF-α

Codonopsis (Codonopsis pilosula (Franch.) Nannf.) is a common medicinal and edible plant with a variety of biological functions, including enhancing gastrointestinal motility, anti-inflammatory, antioxidant, regulating glucose and lipid metabolism, regulating immune response and anti-tumor. Aspirin is a widely used clinical drug with multiple therapeutic effects. However, long-term use of aspirin could also bring some side effects, including gastrointestinal discomfort, liver and kidney damage, asthma, and allergies. The present study is aimed to investigate the effect of Codonopsis decoction on aspirin-induced gastric injuries. Cell proliferation was evaluated by MTT assay and cell apoptosis was investigated by flow cytometry. Gastric mucosal injury index and HE staining were used to evaluate the pathological changes of gastric tissue. The levels of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and transforming growth factor-α (TGF-α) in GES-1 cells culture supernatant were detected by ELISA kits. The levels of cyclooxygenase (COX), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and TGF-α in gastric tissue and serum were detected by ELISA kits. Results showed that Codonopsis decoction significantly attenuated the aspirin-induced cytotoxicity and apoptosis of GES-1 cells. Codonopsis decoction significantly increased the levels of COX-1, COX-2 and TGF-α in GES-1 cells treated by aspirin. Codonopsis decoction could also increase the expression levels of COX and TGF-α in vivo . Codonopsis decoction significantly reduced the levels of TNF-α and IL-6 both in vivo and in vitro . In addition, Codonopsis decoction could increase the daily weight gain of rats taking aspirin, and significantly reduce the gastric tissue damage and gastric mucosal injury index. These data indicated that Codonopsis decoction may possess potential to reduce the side effects caused by oral aspirin and may help the safe use of aspirin.


Introduction
Aspirin is the first non-steroidal drug used clinically in humans with a wide range of pharmacological effects [1]. Because aspirin has the effect of anti-thrombosis in the body, it is widely used in the treatment of cardiovascular and cerebrovascular diseases [2,3]. However, long-term use of aspirin in the process of treating cardiovascular and cerebrovascular diseases can cause gastric mucosal damage, gastrointestinal dysfunction, and eventually show side effects such as nausea, vomiting and gastrointestinal bleeding [4]. TGF-α and COX are important molecules for gastric tissue damage repair. In the process of gastric mucosal injury repair, TGF-α molecules can promote the migration and proliferation of ulcer border cells, promote the regeneration of gastric epithelial tissue, and promote the formation of granulation tissue and angiogenesis [5]. COX is a catalytic enzyme for the metabolism of arachidonic acid. COX-1 is a physiological enzyme that catalyzes the synthesis of prostaglandin (PG) involved in normal physiological functions, and maintains the integrity of the gastrointestinal mucosa [6].
COX-2 is an inducible enzyme produced rapidly by stimulation, which can induce the synthesis of PG in inflammation sites and participate in damage. The metabolite PG compounds have an important protective effect on gastric mucosa [7]. Previous studies showed that aspirin cause mucosal damage, ulceration and ulcer complication throughout the gastrointestinal tract by inhibiting PG synthesis [8]. As important inflammatory cytokines TNF-α and IL-6, which participate in the process of gastric mucosal damage [9].
Codonopsis is a commonly used Chinese herbal medicine and is also used as a food ingredient. Traditional Chinese medical theory believes that Codonopsis can be used to treat syndrome of qi deficiency of spleen and lung, syndrome of anorexia, and syndrome of palpitation [10]. Pharmacological studies showed that Codonopsis extracts exhibit a wide range of pharmacological activities, including gastrointestinal function regulation, endocrine function regulation, immune function regulation, hematopoietic function improvement, cardiovascular protection, anti-aging and antioxidant, and it has not been found obvious toxicity or side effects [11][12][13]. However, the effect of Codonopsis decoction on aspirin-induced gastric mucosal injury has not been reported yet.
This study will investigate the relieving effect and mechanism of Codonopsis decoction on gastric mucosal injury induced by oral aspirin in rats.

Preparation of Codonopsis Decoction
The Codonopsis used in this study is the dried root of Codonopsis pilosula, a plant of Campanulaceae. To prepare Codonopsis decoction, slices of Codonopsis (Tongrentang, Beijing, China) weighing 300 g were soaked in 1 L distilled water for 25 min, then heated and boiled for 30 min. Then the decoction was filtered with 3 layers of gauze and centrifuged at 3000 rpm/min for 20 min.
Finally, the supernatants were collected and concentrated to 300 mL using a rotary evaporator.

MTT Assay
MTT assay was used to examine the effects of Codonopsis decoction on the viability and proliferation of GES-1 cells. Cells were seeded in 96-well plates at 5x10 4  mg/mL aspirin +10 mg/mL OME), aspirin injury group (3.3 mg/ mL) and Codonopsis decoction groups (3.3 mg/mL aspirin + 0.05, 0.1 and 0.2 mg/mL Codonopsis decoction) [14]. Cells in the groups were treated with aspirin or Codonopsis decoction subsequently cultured at 37˚C for 12 and 24 h. All experiments were repeated 3 times.

Animals and Model
Male Sprague Dawley rats weighing 200±20 g were selected to establish a gastric mucosal injury model. First, the rats were weighed and fasted for 12 h. Subsequently, rats were given 60 mg aspirin dissolved in 0.5% carboxymethyl cellulose solution (CMC) for 7 consecutive days.

Grouping Situation
The rats used in the study were randomly divided into 5 groups (Table 1): control group (rats were given 0.5% CMC for 7 d followed by distilled water for another 7 d), aspirin group (rats were given 300 mg/kg aspirin for 7 d followed by distilled water for another 7 d), positive control group (rats were given 300 mg/kg aspirin followed by 15 mg/kg OME for another 7 d), low-dose Codonopsis decoction group (rats were given 300 mg/kg aspirin followed by Codonopsis decoction for another 7 d), medium-dose Codonopsis group (rats were given 300 mg/kg aspirin followed by Codonopsis decoction for another 7 d), and a high-dose Codonopsis group (rats were given 300 mg/kg aspirin followed by Codonopsis decoction for another 7 d). Table 1: Treatment of rats in each group.

Body Weight
The body weight of rats in each group of were respectively weighed at 0 d, 7 d and 14 d. And the daily weight gain of each group was calculated.

Anatomical Lesion
Rats in each group were anesthetized and sacrificed at 14 d. Then stomachs were opened along the greater curvature and washed with saline. According to a previous report, the number and severity of gastric lesions were assessed for overall pathology based on a 0-5 scoring system: [15] 0 points means no lesions, 1 point means small round hemorrhagic lesions, 2 points means that the diameter of the lesion is less than 2 mm, 3 points means that the diameter of the lesion is 2-3 mm, 4 points means that the diameter of the lesion is 3-4 mm, 5 points means that the diameter of the diseased area is greater than 4mm. When the diameter is greater than 1 mm, multiply the score by 2. The average score represents the gastric mucosal injury index (UI). The damage inhibition rate is calculated by the formula: (1-UI Codonopsis /UI Model )×100%.

HE Staining
The lesions of the rat stomach were selected and fixed in 4% paraformaldehyde for 24h. HE staining was performed according to the reported method [16]. The pictures was taken using a microscope (Olympus Corporation, Tokyo, Japan).

Statistical analysis
Results were expressed as means ± standard deviations (SD).
The statistical significance of differences was determined using the Student's t-test or one-way analysis of variance (ANOVA) as appropriate. All the statistical tests were performed using GraphPad Prism software 8.2 (GraphPad software, USA). The p-value < 0.05 was considered statistically significant.

The Effects of Codonopsis Decoction on the Viability of GES-1 Cells
As shown in Figure

Effect of Oral Aspirin on Gastric Mucosa
As shown in Figure 4, the gastric mucosa of the rats in the control group was intact without obvious erosion, bleeding or ulcer formation ( Figure 4A). In contrast, obvious erosions or ulcers were seen on the gastric mucosal of model rats, and the gastric mucosal wrinkles disappeared ( Figure 4B). Results of HE staining showed that the mucosal structure of the gastric tissue of the control group was complete, the mucosal glands were arranged neatly, and there was no bleeding and no inflammatory cell infiltration ( Figure 4C).
In the gastric tissue of the model group, mucosal structural defects, mucosal edema, and interstitial edema can be seen, and there are exudates and necrotic tissue on the surface of the mucosa ( Figure   4D).

Codonopsis Decoction vould Significantly Reduce Gastric Mucosal Injury Index
As shown in Figure 5, compared with the rats in the model group, the gastric mucosal folds of the rats in the low-dose  Table 2).  F. Injury index of rat gastric mucosa.

Codonopsis Decoction Could Significantly Reduce the Stomach Histopathology Induced by Aspirin
As shown in Figure 6, the pathological section of the model group showed that the surface mucosa was damaged, superficial erosion and some necrotic tissue, the boundary between the mucosa and submucosa was blurred, interstitial edema and

Effect of Codonopsis Decoction on Body Weight of Rats
The average daily gain of rats in the model group was

Effect of Codonopsis Decoction on Levels of COX, TNF-α, IL-6 and TGF-α in Serum and Stomach Tissue
As shown in Figure 8, the levels of COX and TGF-α in serum and gastric tissues of rats taking aspirin were significantly lower than those in the control group (P<0.01, Figures 8A-8B). After administration of Codonopsis decoction and OME, the levels of COX and TGF-α in serum and gastric tissue of rats increased significantly.
Compared with the control group, the levels of TNF-α and IL-6 were significantly increased following treatment with aspirin (P<0.01, Figures 8C-8D). However, after administration of Codonopsis decoction and OME, the levels of TNF-α and IL-6 in serum and stomach tissue of rats were decreased significantly.

Discussion
Codonopsis is a classical traditional Chinese herbal, sometimes used as a substitute for Ginseng to widely replenish Qi deficiency, strengthen the immune system, enhance the body's metabolism, improve poor GI function, alleviate gastric ulcers, and improve appetite [17,18]. The in vivo study investigated the alleviating effect of Codonopsis decoction on gastric mucosal injury in rats caused by oral aspirin.
Results show that oral aspirin can induce obvious gastric mucosal damage in rats, which is in accordance to previous studies [19][20][21].
At the same time, after oral administration of aspirin, the daily gain of rats was significantly reduced, the expression levels of COX and TGF-α in serum and stomach tissue were significantly reduced, and the expression levels of TNF-α and IL-6 in serum and stomach tissue were significantly increased. However, after oral administration of Codonopsis decoction, the daily gain of model rats increased, the number of gastric mucosal lesions was significantly reduced, and the pathological changes of gastric mucosa were significantly reduced. It shows that Codonopsis decoction can significantly reduce the gastric injury induced by oral aspirin.
TGF-α is a regulatory peptide of the EGF family, also known as "mucosal intact peptide". It is an endogenous protective factor in cell proliferation and promotes a variety of cells by binding to the receptor epidermal growth factor receptor (EGFR) of mitosis [22][23][24]. TGF-α plays an important role in maintaining the integrity of the mucosa, can regulate the proliferation of gastric mucosal epithelial cells, and participate in the regulation of mucosal epithelial renewal and repair after injury [25,26]. As a commonly used drug, aspirin has antipyretic and analgesic, anti-inflammatory and anti-rheumatic effects [27]. The main mechanism of aspirin anti-inflammatory is to inhibit the synthesis of prostaglandin by inhibiting COX. However, because aspirin does not have the characteristics of targeting, oral aspirin will also inhibit the level of COX in the gastric mucosa [28][29][30]. COX is a key enzyme for the formation of PG, and PG has a protective effect on gastric tissue [31]. This study found that the concentrations of COX in the gastric tissue and serum of rats after oral administration of aspirin were significantly reduced (P<0.01). This result further confirmed the effect of aspirin on the expression of COX. At the same time, we found that the levels of TGF-α in the stomach tissue and serum of rats that were orally taken aspirin were also significantly reduced.
It can be speculated that inhibiting the damage and repair function of gastric mucosal epithelium may be a way for aspirin to cause gastric damage. On the contrary, the levels of TGF-α and COX in serum and stomach tissue of rats that were given Codonopsis decoction after oral administration of aspirin significantly increased. Correspondingly, the gastric mucosal lesions of rats in the Codonopsis decoction group were significantly reduced. TNF-α and IL-6 are cytokines with a wide range of biological effects, which have an important regulatory role in the immune and inflammatory process of the body [32]. These increase in their expression levels usually mean the aggravation of body damage. IL-6 can activate the central nervous system to regulate injury and defense, and at the same time regulate immune response and acute phase response, which is significantly increased in patients with gastric ulcer [33]. TNF-α is a kind of endogenous multidirectional inflammatory cell factors that can activate neutrophils and continue to damage the gastric mucosa, causing ulcers to be difficult to heal [34]. Aspirininduced the high levels of TNF-α and IL-6 in serum and stomach tissue of rats were also significantly attenuated by Codonopsis decoction at all doses. These data suggest that the Codonopsis decoction may relieve aspirin-induced gastric mucosal damage by reducing the inhibitory effect of aspirin on COX and TGF-α, and the high expression levels of TNF-α and IL-6.
Pyrazole drugs are usually used to treat gastric mucosal damage caused by aspirin, but pyrazole drugs can also bring side effects such as hypomagnesemia and skin damage [35,36]. Codonopsis is a traditional Chinese medicine that has been used for hundreds of years, and it is also a plant with the same medicine and food [37].
It is enough to ensure the safety of Codonopsis. It should be noted that more than one hundred compounds have been isolated and identified from Codonopsis, including polysaccharides, alkaloids, lactone compounds, volatile oils, etc. [38]. These compounds endow Codonopsis with a variety of biological effects. It may be one of the ingredients that plays a specific role, or it may be a combination of several ingredients. In addition, the results of this study indicate that Codonopsis decoction has effects on COX, COX-1, COX-2, TNF-α, IL-6 and TGF-α. Combined with the multi-target characteristics of traditional Chinese medicine, [39] we believe that the effect of Codonopsis decoction may be better than that of a single compound.
In summary, the results of the study indicate that Codonopsis decoction may target on COX, COX-1, COX-2, TNF-α, IL-6 and TGF-α to reduce aspirin-induced gastric mucosal injury both in vivo and in vitro. This suggests that Codonopsis may be a potential choice to relieve or eliminate the side effects of aspirin. Of course, more specific works still need to be carried out to clarify the exact pharmacological effects of the different ingredients in Codonopsis decoction on the above aspects.