Effects of Tongluo Jiedu prescription on immune function and oxidative stress in patients with oral cancer

Abstract

BACKGROUND

Oral cancer, which is caused by mucous membrane variation, represents a prevalent malignant tumor in the oral and maxillofacial region, posing a significant threat to patients’ lives and safety. While surgical intervention stands as a cornerstone treatment for oral cancer patients, it carries the risk of incomplete treatment or high rates of postoperative recurrence. Hence, a multifaceted approach incorporating diverse treatment modalities is essential to enhance patient prognosis.

AIM

To analyze the application effect of Tongluo Jiedu prescription as adjuvant therapy and its influence on patient prognosis in patients with oral cancer.

METHODS

Eighty oral cancer patients in our hospital were selected and divided into the observation group and control group by a random number table. The control group was treated with continuous arterial infusion chemotherapy of cisplatin and 5-fluorouracil. The observation group was additionally given Tongluo Jiadu prescription. The inflammatory stress level, peripheral blood T-cell subsets, and immune function of the two groups were subsequently observed. SPSS 21.0 was used for data analysis.

RESULTS

The observation group demonstrated lower levels of interleukin-6 and C-reactive protein, and a higher level of tumor necrosis factor in comparison to the control group. After treatment, the immune function in the observation group was significantly better than in the control group.

CONCLUSION

Tongluo Jiedu prescription can improve the immune function and oxidative stress level of patients with oral cancer and accelerate the recovery process.

Key Words: Tongluo Jiedu prescription, Oral cancer patients, Immune function, Oxidative stress

Core Tip: This study prospectively selected 80 patients with oral cancer, and used collateral detoxification as an auxiliary means for continuous arterial perfusion chemotherapy. Tongluo Jiedu prescription improved the immune function and oxidative stress level of patients with oral cancer and accelerated the recovery process.

INTRODUCTION

Oral cancer is a common malignant tumor in the oral and maxillofacial region caused by mucous membrane variation, and it is a threat to patients' life and safety. Surgery is one of the important treatment methods for patients with oral cancer, but patients still face the risk of incomplete treatment or high recurrence rate after treatment[1]. Therefore, a variety of comprehensive treatment methods should be combined to improve the prognosis of patients. Studies have found that tumors can develop resistance to postoperative adjuvant chemotherapy, and its curative effect is often geometrically related to drug concentration[2]. Therefore, how to improve the concentration of chemotherapy in the target area is the key to improving the curative effect of chemotherapy. Regional continuous arterial infusion chemotherapy can increase the local drug concentration in the tumor and improve the efficacy of chemotherapy for oral cancer[3]. However, long-term chemotherapy also has some toxic side effects, such as bone marrow suppression and gastrointestinal reactions, which can reduce the immune function of patients. In recent years, experience has been gained of traditional Chinese medicine for tumor treatment, which is important for improving the therapeutic effect, reducing the toxic side effects of chemotherapy, and improving the immune function of patients. In traditional Chinese medicine, it is currently believed that oral cancer originates from the internal invasion of toxic pathogens, which belong to the categories of "oral sores", "oral chyme" and "sore selection". Due to the obstruction of the complex veins, the removal of toxic blood stasis and long-term damage to Yin, the treatment should focus on fuzheng dispelling evil, removing blood stasis and clearing up collateral, and clearing heat and detoxification[4]. Based on this, this study adopted collateral detoxification for postoperative oral cancer patients to assist continuous arterial perfusion chemotherapy, to analyze its application as adjuvant therapy and its influence on patient prognosis.

MATERIALS AND METHODS

Research objective

Eighty patients with oral cancer in our hospital were selected by random number table method and divided into the observation group and control group. There was no significant difference (P > 0.05) between the two groups of patients in terms of gender, age, body mass index, and the number of other cancer patients (Table 1).

Table 1 Comparison of baseline data between the observation and control groups.

Variables	Classification/unit	Observation group (n = 40)	Control group (n = 4 0)	t/χ2	P value
Gender	Male	28	27	0.058	0.809
	Female	12	13
Age	yr	53.41 ± 14.67	54.32 ± 11.61	0.308	0.759
BMI	kg/m2	21.45 ± 2.76	21.17 ± 3.92	0.172	0.864
Other cancers	Yes	3	2	0.213	0.644
	No	37	38

BMI: Body mass index.

Inclusion criteria: (1) Patients met the relevant diagnostic criteria for oral cancer in "Guiding Principles for Clinical Research of New Chinese Medicine" and "Stomatology"; (2) Karnofsky Performance Scale > 70; (3) predicted survival time > 6 mo; and (4) no symptoms of distant metastasis.

Exclusion criteria: (1) Abnormal liver and kidney function; (2) other malignant tumors; (3) contraindications to the drugs used in this study, or had other allergic conditions; (4) had received chemotherapy before inclusion in the study; and (5) accompanying serious systemic diseases. Elimination criteria: (1) Patients must terminate treatment due to severe side effects during treatment; (2) patients were willing to quit the study midway; and (3) patients with poor compliance.

Research methods

After the patients' systemic condition recovered well after surgery, the control group was given continuous arterial infusion of 100 mg/m² cisplatin microinjection pump for 24 h, and continuous arterial infusion chemotherapy of 1000 mg/m² 5-fluorouracil for 3 d, with 21 d as a course of treatment. The observation group was additionally given Tongluo Jiadu prescription, which included 24 g astragalus, 30 g dandelion, 15 g Jiu Xiang Xiang, 30 g Serpentine herb, 15 g salvia miltiorrhiza, 15 g coix seed, 15 g bergamot, 15 g sauteed jujube kernel, 15 g cistanche, 10 g paeoniae, 10 g ophiopogon, 10 g licorice, and 10 g turniflora. The above formula was boiled in water once a day, and the juice was 400 mL. Patients should take warm water in the morning and evening, with 21 d as a course of treatment. Both groups were treated for three consecutive courses.

Observation indicators

First, inflammatory stress level: 3 mL fasting venous blood was collected from the patients in the morning before and after treatment, and the serum was centrifugated to detect tumor necrosis factor (TNF)-α level by ELISA, and C-reactive protein (CRP) level by scattering turbidimetric assay. At the same time, 3 mL of fasting venous blood was collected in the morning before treatment and 1 d after surgery, and serum was centrifuged to detect the level of interleukin (IL)-6 by ELISA. Second, peripheral blood T-cell subsets: before and after treatment, 5 mL fasting venous blood was collected in the morning, and the levels of T-cell subsets (CD4⁺, CD8⁺, and CD4⁺/CD8⁺) were measured by flow cytometry. Finally, immune function: 5 mL of fasting venous blood was collected from patients in the morning before and after treatment, and the serum was centrifuged to detect the levels of IgA, IgG and IgM) in the serum by immunoturbidimetric method.

Statistical methods

SPSS 21.0 was used for data analysis. The χ² test was used for comparison among groups, and the data that conformed to a normal distribution were presented as mean ± SD. Independent sample t test was used for comparison between groups, and paired sample t test was used for comparison of patients in the same group before and after treatment. P < 0.05 was considered statistically significant.

RESULTS

Inflammatory stress levels before and after treatment

Serum CRP level increased in the observation and control groups after treatment (Table 2). Further analysis revealed that CRP level was lower in the observation group (27.45 ± 5.92 μg/L) compared with the control group (39.42 ± 7.84 μg/L). There was an increase in serum IL-6 level in both groups; however, it was significantly lower in the observation group (64.23 ± 13.29 μg/L) compared with the control group (72.57 ± 15.23 μg/L). The treatment reduced the decline in TNF-α in the observation group (6.45 ± 1.22 ng/L), compared with the control group (5.12 ± 1.16 ng/L).

Table 2 Comparison of inflammatory stress levels before and after treatment (mean ± SD).

Indicators	Time	Observation group (n = 40)	Control group (n = 40)	t	P value
CRP (μg/L)	Before treatment	8.69 ± 2.14	9.17 ± 2.03	1.029	0.306
	After treatment	27.45 ± 5.92a	39.42 ± 7.84a	7.693	0.000
IL-6 (μg/L)	Before treatment	51.47 ± 11.64	50.03 ± 12.45	0.534	0.595
	After treatment	64.23 ± 13.29a	72.57 ± 15.23a	2.610	0.011
TNF-α (ng/L)	Before treatment	9.10 ± 1.87	8.84 ± 1.81	0.632	0.529
	After treatment	6.45 ± 1.22a	5.12 ± 1.16a	4.997	0.000

^aP < 0.05, compared with before treatment. CRP: C-reactive protein; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor alpha.

T-cell subsets before and after treatment

After treatment, there was a notable reduction in the CD4⁺ and CD4⁺/CD8⁺ levels in the peripheral blood of both groups (Table 3). However, the observation group (9.62 ± 2.21, 2.54 ± 0.55, respectively) exhibited significantly elevated levels compared with the control group (8.86 ± 1.84, 1.97 ± 0.47, respectively). Additionally, the CD8⁺ levels increased post-treatment, with the observation group showing lower levels (4.45 ± 0.76) than the control group (5.17 ± 0.92).

Table 3 Comparison of T-cell subsets before and after treatment (mean ± SD).

Indicators	Time	Observation group (n = 40)	Control group (n = 40)	t	P value
CD4+ (%)	Before treatment	12.15 ± 2.41	11.63 ± 2.32	0.983	0.329
	After treatment	9.62 ± 2.21	8.86 ± 1.84	1.671	0.099
CD8+ (%)	Before treatment	3.41 ± 0.66	3.33 ± 0.64	0.550	0.584
	After treatment	4.45 ± 0.76a	5.17 ± 0.92a	3.816	0.000
CD4+/CD8+	Before treatment	3.52 ± 0.82	3.46 ± 0.75	0.341	0.734
	After treatment	2.54 ± 0.55a	1.97 ± 0.47a	4.983	0.000

^aP < 0.05, compared with before treatment.

Immunoglobulin levels before and after treatment

Serum IgA, IgG and IgM levels in the observation group and control group (1.92 ± 0.41, 5.51 ± 1.21 and 0.52 ± 0.13, respectively) were significantly lower than they were prior to treatment, with the levels in the observation group being higher (2.46 ± 0.40, 7.64 ± 1.49 and 0.71 ± 0.17, respectively) (Table 4).

Table 4 Comparison of immunoglobulin levels before and after treatment (mean ± SD, g/L).

Indicators	Time	Observation group (n = 40)	Control group (n = 40)	t	P value
IgA	Before treatment	2.81 ± 0.59	2.72 ± 0.55	0.706	0.483
	After treatment	2.46 ± 0.40a	1.92 ± 0.41a	5.962	0.000
IgG	Before treatment	10.57 ± 2.34	11.02 ± 2.28	0.871	0.386
	After treatment	7.64 ± 1.49a	5.51 ± 1.21a	7.018	0.000
IgM	Before treatment	1.02 ± 0.24	1.06 ± 0.22	0.777	0.440
	After treatment	0.71 ± 0.17a	0.52 ± 0.13a	5.615	0.000

^aP < 0.05, compared with before treatment.

DISCUSSION

At present, postoperative chemotherapy is often given to patients to treat cancer. Although it can effectively kill cancer cells, it can also damage the patients' autoimmune function. In the five viscera and six organs, kidneys can stabilize and regulate the reproductive and immune functions of the body, which is the foundation of immunity. Spleen is the material basis of immune activity, which can enhance the effect of immune function[5]. The collaterals and detoxification method adopted in this study can regulate and replenish the five Zang organs, improve immunity and promote recovery of the patient. Modern pharmacology has confirmed that astragalus has the antitumor effect of promoting apoptosis of cancer cells, and can also enhance immune function, enhance the phagocytosis function of the reticuloendothelial system, and improve the lymphocyte conversion rate, and the total saponin of astragalus can effectively clear the oxygen free radicals and slow down the oxidative stress reaction[6]. Drugs such as turgeon and salvia miltiorrhiza have the effect of removing blood stasis, which can improve hemodynamics and promote absorption of local inflammation[7]. Nine fragrant worms have anticancer functions[8]. In view of the pharmacological mechanism of Tongluo Jiedu prescription, patients with oral cancer in our hospital were prospectively selected to receive continuous arterial perfusion chemotherapy assisted by Tongluo Jiedu prescription after surgery to observe the effects on immune function and oxidative stress response. If the curative effect is accurate, it can effectively improve the patients' condition, promote disease outcome, improve postoperative quality of life and prevent postoperative recurrence. The innovations of this study were as follows. (1) The main treatment methods for patients with oral cancer are resection and chemotherapy. To date, there have been no relevant studies on the clinical efficacy of Tongluo Jiedu prescription assistance of continuous arterial infusion chemotherapy in patients with oral cancer and its influence on immune function and oxidative stress. (2) Tongluo Jiedu prescription may improve the immune function of patients, reduce oxygen free radicals caused by treatment and disease, relieve oxidative stress reaction, and improve the outcome of disease.

As a common oral and maxillofacial malignant tumor, the incidence of oral cancer ranks second after head and neck tumors, and most of its pathological grades are intermediate or advanced differentiated cancers, and regional lymph node metastasis readily occurs, posing a serious threat to the life and safety of patients. Therapeutic excision is an effective treatment for oral cancer, but there are often considerable risks, such as postoperative recurrence, incomplete excision or other serious complications[9]. Although chemotherapy has obvious antitumor effects, long-term use causes serious damage to the immune function of patients. In recent years, studies have found that oxidative stress injury is closely related to the occurrence and development of oral cancer, and treatment and chemotherapy can lead to the generation of free radicals in large quantities, exacerbating oxidative stress of patients[10].

Oxidative stress occurs when a large number of free radicals build up in the cell and the cellular endogenous antioxidant system is unable to effectively remove them. Oxidative stress is a state in which the oxidation and antioxidant systems in the body are completely out of balance, which may promote the generation and development of various diseases. In the process of radical treatment of patients, the removal of diseased lymph nodes and the tissue incision generated in the process cause blood circulation disorders. In addition, due to stress damage during treatment, a large number of free radicals are produced in the body, leading to lipid peroxidation and mass generation of related peroxidation products such as malondialdehyde (MDA). At the same time, production of antioxidants such as superoxide dismutase (SOD) is inhibited. In addition, factors such as immunosuppression, nutrient intake disorders and the use of antitumor drugs cause production of a large number of free radicals in patients after radical surgery, resulting in a state of oxidative stress. If this state cannot be effectively improved, the postoperative rehabilitation of patients is also be affected. Studies have shown that SOD in saliva and blood of patients with oral cancer is significantly decreased but MDA is significantly increased, which proves that oxidative stress is related to the occurrence of oral cancer[11]. Peripheral stimuli such as smoking, chewing areca nut and drinking alcohol are high risk factors for oral cancer. Oxidative stress can lead to DNA damage and promote the metastasis and spread of cancer cells, and in this way participate in the occurrence and development of oral cancer. At the same time, it has been found that oral epithelial cancer cells cultured with tobacco impregnation solution increase the production of free radicals in the cells, and oxidize and damage nuclear DNA, leading to initiation of the apoptotic pathway[12]. In addition, human oral squamous cell carcinoma cells showed an increase in ROS after being treated with areca extract. Studies have shown that ROS can promote the biological behavior changes of oral cancer cells, induce epithelial–mesenchymal transformation of oral cancer cells, and further develop metastasis and proliferation of cancer cells[13].

The use of traditional Chinese medicine to treat various diseases has become a clinical trend. Oral cancer is considered to belong to the categories of "oral sores", "oral chyme" and "sore selection" in traditional Chinese medicine[14]. The different parts of the mouth are classified as the five Zang organs. The gums belong to the kidneys, the tongue is the heart seedling, the cheeks belong to the liver, the palate and the lips belong to the spleen and are connected with the stomach and lungs, the heart, the liver, the lung and the stomach belong to the viscera of making fire, the liver and the stomach are the viscera of regulating the qi, if the qi is abundant, it is the fire, and the spleen and stomach are the viscera of producing phlegm and dampness. Therefore, in traditional Chinese medicine, it is believed that the incidence of oral cancer belongs to the hot poison and phlegm dampness stagnation internal invasion of the viscera, its disease development and phlegm, and blood stasis are related. Therefore, the location of oral cancer is related to the choroid, and the basic treatment method is collaterals and detoxification. Among the drugs used in the method of collaterals and detoxification, jujube kernel can nourish the heart, and astragalus can invigorate the spleen and kidney, replenish the heart fire to produce stomach soil, replenish the qi of liver and gallbladder and soothe the mind[15]. Cistanche moistens and nourishes the kidney, nourishes the innate spleen and stomach, and the ophiopogon is good for nourishing the stomach, heart and lung Yin[16]. The combination of the four medicines can replenish the five viscera and thus promote the recovery of the body. Salvia miltiorrhiza and blood line Qi can nourish blood and promote blood circulation; Bergamot can awake the spleen and regulate the qi and ease the liver; Yujin Huoxue analgesic, qi Jieyu. The combination of three medicines can effectively promote qi and remove blood stasis. Dandelion and hedyoglossia can clear away canopy heat and heat toxicity. Paeony slows pain relief, soft liver and stomach, collect Yin and nourish blood. The nine fragrant worm dregs liver depression, helps kidney Yang, wake up temper; Liquorice Yiqi and can reconcile various drugs, the whole prescription of all drugs played together to Fuzheng Tongluo detoxification. Studies have found that astragalus saponins have good antioxidant and oxygen free radical scavenging effects[17]. Astragalus saponins are the main active components of Astragalus, and their pharmacological effects may be related to intracellular signal transduction, which can reduce the activation of the p38MAPK signaling pathway to improve apoptosis induced by oxidative stress[18]. Currently, oral cancer is still one of the malignant diseases in the department of stomatology. How to better improve the condition of patients and improve the quality of life is a hot topic in clinical research.

The treatment of oral cancer is still based on local or systemic chemotherapy[19]. However, the postoperative recurrence rate and metastasis rate are often high, and the trauma is serious. There is no such thing as oral cancer in ancient Chinese medicine books, so oral cancer can be divided into the categories of "oral sores", "oral chyme" and "sore selection" in Chinese medicine[20]. It is related to the dysfunction of viscera (spleen, kidney, stomach, heart) and other organs, and is mostly caused by Yin deficiency and blood stasis, deficiency of Yin and blood of liver and kidney, weakness of hormone body, and dysfunction of spleen transport. "The heart meridian is hot, the heart fire on the inflammation, the extreme heat sores; Blood is not transported, spleen and stomach disorders, phlegm and dampness endogenous"; "Kidney accumulation of essence, pulp, for the birth of this; Spleen is the source of biochemistry, is the acquired; The qi will be weak when the evil is pooled together." Therefore, removing blood stasis and detoxifying, strengthening spleen and kidney, and strengthening Zhengzheng and Guzhen are the common therapeutic principles used in TCM to treat cancer.

Tongluo detoxification formula in dandelion: return to the liver, stomach meridian[21]. Clear heat, detoxify and dehumidify. Snake grass: into the stomach, large intestine, small intestine. Clear heat, dampness, detoxification, carbuncle. Astragalus: return to the spleen, lung meridian. Qi Sheng Yang, Yi Wei Gu table, Tuo Tuo Sheng muscle, Li Shui detumescence. Nine fragrant worm: spleen, liver, kidney meridian. Qi analgesic, warm kidney and Yang. Coix seed: spleen, stomach, lung meridian. Li water infiltration, spleen, remove Bi, clear heat and discharge pus. Salvia miltiorrhiza: heart, liver meridian. Promote blood stasis, analgesia, clear the heart and eliminate boredom, cold blood carbuncle. Bergamot: liver, spleen, lung meridian. Regulate the liver Qi, and stomach phlegm. Sauted jujube kernel: return to liver, gallbladder, heart meridian. Nourishing the heart and nourishing the liver, Ningxin calming the mind, collecting sweat, Shengjin. Cistanche: return to kidney, large intestine meridian. Tonifying the kidney and helping the Yang, moistening the bowel. Maidong: Heart, lung, stomach meridian. Nourish Yin Shengjin, moisten lung cough. White peony root: return to liver and spleen meridian. Nourishing blood and regulating meridian, collecting Yin and antiperspirant, soothing liver and relieving liver Yang. Yu Jin: The heart, liver and gallbladder classics. Activating blood to relieve pain, Qi Jieyu, cool serum heart, Li Dan regressive yellow. Licorice: heart, lung, spleen, stomach meridian. Tonifying spleen and Qi, clearing heat and detoxifying, expectorating cough, relieving pain and relieving pain, harmonizing various medicines[22]. The effect of the whole prescription to play together in Fuzheng, collaterals and detoxification[23].

The stress response caused by surgical trauma has different degrees of influence on the expression of inflammatory factors and immune function of patients, but immunosuppression may lead to the increase of postoperative infection. CRP is an acute response protein synthesized by IL-6, and the expression levels of both in serum are closely related to the degree of injury. The severity of treatment trauma is related to the concentration of CRP and IL-6 in serum, and the higher the concentration of CRP and IL-6, the more severe the trauma, while TNF-α is considered to be an inflammatory factor with antitumor effects[24]. In this study, serum CRP and IL-6 levels were increased and TNF-α levels were decreased in the two groups 3 d after surgery compared with before treatment, and the range of change was small in the observation group, indicating that the treatment of Tongluo Jiedu prescription had little effect on the inflammatory stress response of patients with oral cancer. The immune response of patients with oral cancer is mainly mediated by T cells[25]. CD4⁺ cells are auxiliary and inductor T cells, which release a large number of cytokines after activation to enhance the antitumor effect. CD8⁺ cells, as cytotoxic T cells, have the function of eliminating viruses, and the level of CD4⁺/CD8⁺ can reflect immune function. Significantly reducing its ratio is beneficial to proliferative tumor cells. IgG, IgA, IgM and other immunoglobulins are involved in humoral immunity, and their expression in serum can reflect immune function[26]. The results of this study showed that the levels of serum IgA, IgG and IgM and the levels of CD4⁺ and CD4⁺/CD8⁺ in peripheral blood of the two groups were decreased 3 d after surgery compared with before treatment, and the levels of related substances in the observation group were higher than those in the control group, indicating that the postoperative immune function of patients with oral cancer was indeed decreased by the influence of treatment trauma. However, Tongluo Jiedu prescription has little effect on immune function, which is beneficial to postoperative recovery of patients.

CONCLUSION

Tongluo Jiedu prescription improves the immune function and oxidative stress level of patients with oral cancer, and accelerate their recovery process.