Sufentanil postoperative analgesia reduce the increase of T helper 17 (Th17) cells and FoxP3⁺ regulatory T (Treg) cells in rat hepatocellular carcinoma surgical model: A randomised animal study

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, characteristic of relatively poor overall survival and increasing morbidity and mortality, which is reportedly the third cancer-related mortality worldwide [1, 2]. Surgery-related pain and opioid analgesics are factors known to adversely affect the anti-tumor immune defenses which may promote tumor growth and metastasis [3]. In view of the growing interest in the immune system in control of neoplasia, further efforts toward the discovery of a good analgesia agent for postoperative pain treatment with a reduced impaction on immunity are urgently needed.

The helper T cells were mainly divided into T helper 1(Th1), Th2, Th9, Th17, Th21, T follicular helper (Thf) and regulatory T (Treg) cells according to the function and phenotype [4, 5]. Among them, Th1, Th2, Th17 and Treg cells are more concerned in tumor immunity. Th17 cells could increase tumor progression by activating angiogenesis and immunosuppressive activities [6, 7]. Treg cells might inhibit the tumor-specific T cell-mediated immune response and have been observed increased quantity in tumor tissues or peripheral blood of patients or animal models with gastric cancer [8], ovarian cancer [9], breast cancer [10] and hepatocellular carcinoma [11].

Immune cells express appropriate receptors such as the μ receptor and toll-like receptor. Opioids modulate the immune system by binding to the μ receptor [12]. Sufentanil has a higher affinity to μ1-opioid receptor which has the closest relationship with analgesia than morphine, but the selectivity for binding to μ2 receptor is opposite which is related to adverse effects such as nausea, vomiting, respiratory depression, urinary retention, and itching, so sufentanil has stronger analgesic effect than morphine, and adverse effects are weaker than morphine [13].

The results of opioid-induced immunomodulation are conflicting in experimental and human studies. Previous studies manifested that morphine could decrease the expressions of peripheral T lymphocytes (CD3⁺, CD4⁺, CD8⁺) and natural killer cells (CD3⁺, CD56⁺) in vivo [14] and could increase the ratio of CD4⁺/CD8⁺ T cells and Treg populations in vitro [15]. The Epidural postoperative analgesia with ropivacaine plus sufentanil significantly decreased Blymphocytes, T-helper cells and Natural killer cells compared with patient-controlled IV analgesia (PCIA) with morphine in patients after major spine surgery [16]. However, little or nothing is known concerning the effect of sufentanil postoperative analgesia on Th17 and Treg cells. The primary purpose of this study was to observe the effects of sufentanil and morphine postoperative analgesia on immunity through analysis of CD4⁺/CD8⁺ ratio, proportion of Th1, Th2, Th17 and Treg cells using flow cytometry, and the secondary target was liver function changes and mortality in HCC rats undergoing left hepatolobectomy.

The food and water consumption, body weight, locomotor activity and the pain threshold in each group—The food consumption, water consumption, locomotor activity and MPT of Group C decline to a significantly lower degree than those of Group S and M on d1, d2, d3(P < 0.05). The food consumption, water consumption, locomotor activity and pain threshold of Group S were similar with that of Group M at each time point. There were no significant differences of the body weight between the three groups (Fig. 1).

CD4⁺/CD8⁺ratio in blood of each group—Fig. 2. A shows the flow cytometric analysis of CD4⁺ and CD8⁺ cells. Figure 2. B shows the statistical analysis of CD4⁺/CD8⁺ ratio. The CD4⁺/CD8⁺ ratio of Group S and Group M were significantly higher than that of Group C on d3 and d7 (P < 0.05). The CD4+/CD8+ ratio of Group S was significantly higher than that of Group M on d3 (P < 0.05).

The proportion of Th1 and Th2 cellsin blood of each group—Fig. 3. A and C shows the flow cytometric analysis of Th1 (CD4⁺IFN-γ⁺) and Th2 (CD4⁺IL4⁺) cells. Figure 3. B and D shows the statistical analysis of Th1 and Th2 cells. The proportion of Th1 cells of Group S and Group M were significantly higher than that of Group C on d3 and d7 (P < 0.05, Fig. 3. B). The proportion of Th2 cells of Group S and Group M were significantly lower than that of Group C on d3 and d7 (P < 0.05, Fig. 3. D). There were no statistically significant differences in proportion of Th1 and Th2 cells between Group S and Group M on d3 and d7 (Fig. 3. B and D).

The proportion Th17 and Treg cells in blood of each group—Fig. 4. A and C shows the flow cytometric analysis of Th17 (CD4⁺IL17-A⁺) cells and Treg (CD4⁺Foxp3⁺) cells. Figure 4. B and D shows the statistical analysis of Th17 and Treg cells. Rats showed lower proportion of Th17 and Treg cells in Group S and Group M than group C (p < 0.05, Fig. 4. B and D). There were no statistically significant differences in proportion of Th17 cells between Group S and Group M on d3 and d7, however, the proportion of Treg cells in Group S was significantly lower in comparison to Group M on d7 (p < 0.05, Fig. 4. D).

The liver function in each group after surgery—A significant increase of ALT and AST levels was observed in group C in comparison to Group S and Group M on d7(p < 0.05). But no statistically significant difference was observed between Group S and Group M (Fig. 5. A and B).

The survival situation—Though we did not find statistically significant differences in mortality rate between postoperative analgesia rats and without analgesia rats (P = 0.245, Fisher’s Exact Test). We did observe that two rats of Group C died respectively on fourth and fifth day after surgery, one rat of Group M died on sixth day after surgery, and no rat died in Group S.

This study found that sufentanil and morphine postoperative analgesia rats have higher CD4⁺/CD8⁺ ratio, Th1 cells level while lower Th2, Th17 and Treg cells levels compared with that without postoperative analgesia. Sufentanil postoperative analgesia rats have higher CD4⁺/CD8⁺ratio on the third day after surgery while lower Treg cells level on the 7th day after surgery in comparison to morphine postoperative analgesia rats.

Acute postoperative pain can activate the hypothalamic–pituitary–adrenal axis, affect metabolism and cause neuroendocrine changes, which are strongly associated with postoperative outcome [12, 22, 23]. Postoperative pain relief can reduce surgery-associated cardiac, pulmonary, metabolic complications, and improve immune status which may improve the postoperative outcome [24].

There is such a view that the interaction between CD4⁺ and CD8⁺ T lymphocyte mediates the control of tumor growth [25]. In a clinical study, the 5-year survival rate of cervical cancer patients with high CD4⁺/ CD8⁺ ratio was higher than that of patients with low CD4⁺/ CD8⁺ ratio, increasing the CD4⁺/ CD8⁺ ratio can slow the progression of cervical cancer and improve its prognosis [26]. It is generally believed that Th1 enhances tumor immune surveillance of tumor while Th2 associated with the tumor immune evasion can suppress the function of Th1 cells [27]. Th17 cells in peripheral blood are positively correlated with the progression of liver cancer [28]. Treg cells play a vital role in maintaining immunological homeostasis and exert major immunosuppressive activity [29]. A recent study has indicated that the percentages of CD4⁺CD25⁺FOXP3⁺Treg cells and CD4⁺IL-17⁺Th17 cells were significantly higher in HCC patients than in the healthy individuals; Moreover, the increased percentages of Treg and Th17 cells were closely related to the tumor stage and tumor size of HCC [11]. Most published research have found that post-operative opioids inhibit cell-mediated immunity and promote tumor metastasis for both human and mouse [3]. Some patients choose to tolerate pain because of concerns about the immunosuppressive effect of analgesics. Is this appropriate? In our study, the CD4⁺/CD8⁺ratio, proportion of Th1 cells were obviously higher while proportion of Th2, Th17 and Treg cells were significantly lower in group S and group M compared with group C. Therefore, it seems that sufentanil and morphine postoperative analgesia can alleviate the immunosuppressive effect of HCC surgery and postoperative pain and is more conducive to postoperative recovery than tolerating pain.

In terms of analgesic effect, both of sufentanil and morphine can control postoperative pain. Which is better for sufentanil and morphine postoperative analgesia? Previous studies have demonstrated that morphine affect the signal transportation of activated T cells, thereby inhibiting T-cell activation. Morphine increases the ratio of CD4⁺/CD8⁺and Treg cells populations [15, 30, 31], shifts the balance of Th1/Th2 cells toward Th2 cells [32, 33], while in vivo studies, the ratio of CD4⁺/CD8⁺cells, the proportion of Th1 and Th17 T cell were not changed with the administration of morphine [30]. Sufentanil increased the quantity of the Tregs to a greater degree than fentanyl when the culturing was conducted in vitro, while there was no significant difference between them in vivo [34]. In a clinical trial, total CD3⁺, CD4⁺, CD8⁺ cells and the ratio of CD4⁺/CD8⁺ cells in the sufentanil group were significant higher than that in the remifentanil group [35]. There are few direct comparative studies which involved in the effects of sufentanil and morphine on immunity. In our study, sufentanil and morphine have a similar effect on Th1, Th2, Th17 frequencies. Yet, the ratio of CD4⁺/CD8⁺ on d3 after surgery and the ratio of Treg cells on d7 after surgery in Group S is obviously less than that of Group S. This results indicate that sufentanil’s inhibition on CD4⁺ cells is lighter than morphine, but this inhibition may disappear with the withdrawal of the drugs. While the inhibition of sufentanil on Treg cells is less than that of morphine, but the inhibition of Treg cells may be manifested later. Recently, increasing studies have shown that there is a close positive correlation between recurrence and metastasis to the inhibition of immune system [36, 37]. Therefore, it seems that sufentanil is superior to morphine for postoperative anelgesia. The findings of the presented study provide help for the selection of postoperative analgesic drugs in clinic.

AST and ALT are important enzymes which represent liver cells function [38]. AST and ALT evidently increased can reflect severe liver cells necrosis. AST/ALT were the independent risk factors of overall survival [39]. Previous retrospective study indicated that hepatic cancer patients who underwent hepatectomy with higher ALT level had shorter mean recurrent interval than patients with lower ALT level [40]. In our study, we found that postoperative plasma ALT and AST values on the seventh day in Group S and Group M were significantly lower than in Group C. This suggests that postoperative analgesia can prevent liver function damage in HCC rats accepted hepatectomy.

There are some shortcomings in our experiment. First, we only measured the number of some T cell subsets without measuring important immune factors, such as TGF-beta, IL-6, IFN-gamma. Second, these rats’ long-term survival, metastasis rates were not observed. We will evaluate these results in the future.

The current results have shown that sufentanil and morphine postoperative analgesia in HCC rats accepted hepatectomy can relieve postoperative pain, promote the recovery of liver function after surgery, alleviate the immunosuppressive effect of pain. Furthermore, Sufentanil postoperative analgesia is better than morphine resulted by the differences of CD4⁺/CD8⁺ ratio and Treg cells level after surgery.