Tetrahydropalmatinee alleviates diabetic neuropathic pain by inhibiting the inammation via p38-MAPK signaling pathway in microglia

Object: Exploring the effect of Tetrahydropalmatine (THP) on diabetic neuropathic pain (DNP) and its possible mechanism. Methods: The type 2 diabetic (T2DM) rat models were prepared by high-fat and high-sugar feeding combined with a single small-dose intraperitoneal injection of streptozotocin (STZ). When the mechanical withdrawal threshold (MWT) and the thermal withdrawal latency (TWL) of T2DM model rats decreased to less than 85% which were judged as DNP-bearing rats. After treatment with or without THP, the protein expression of hypertonic glycerol reactive kinase (p38), phosphorylated hypertonic glycerol-responsive kinase (p-p38) and OX42 (a specic marker of microglia) were detected by Western Blot and and the mRNA content of p38 and OX42 were detected by qRT-PCR. The expression of pro-inammatory factors IL-1β, IL-6, TNF-α, as well as chemotactic factors and their receptors including CXCL1, CXCR2, CCL2 and CCR2 in spinal tissues were detected by ELISA. Serum FINS and GSP content were also detected by ELISA. Double-label immunouorescence were used to observe the expression of OX42 and p-p38 in the spinal dorsal horn. Results: Results showed that THP inhibited microglial activation of spinal in DNP rats. And after THP intervention, the MWT and TWL of DNP rats decreased, the expression of p38, p-p38 and OX42 in the spinal cord tissues of rats was signicantly reduced while the mRNA of p38 and OX42 also reduced. The expression of IL-1β, IL-6, TNF-α, CXCL1, CXCR2, CCL2 and CCR2 in the spinal cord tissues of rats was signicantly reduced (P < 0.01). At the same time, THP signicantly proved FINS, but did not affect FBG and GSP in DNP rats. be achieved by inhibiting the inammatory response caused by the activation of microglia mediated by the p38-MAPK signaling pathway.


Introduction
According to the latest International Diabetes Federation (IDF) investgation in 2019, about 6% of the world's population is suffering from diabetes, and this number is increasing year by year, the proportion may reach 10% of the total population by 2045 [1]. What's More, about 20%-30% diabetic patients are sufferring from DNP. The clinical features of DNP are spontaneous pain, paresthesia and hyperalgesia, which seriously affects the physical and mental health of patients [2]. Although the underlying mechanism of DNP is still unclear, there's a growing body of evidence suggestis that the activation of spinal microglia may plays an important role in the occurrence and development of DNP [3,4].
Microglia, the intrinsic macrophages of the central nervous system, are activated in the spinal cord in neuropathic or in ammatory pain (5). It also mediates neuroin ammations and plays an important role in the occurrence and development of DNP [5]. Studies have shown that the spinal cord microglia are activated in STZ-induced diabetic rats [6,7]. The activation of microglia is accompanied by the release of cytokines and the activation of the p38-MAPK signaling pathway, which is associated with hypersensitivity of in ammatory pain [8]. The phosphorylation activation of p38 leads to the production of pro-in ammatory mediators,such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and interleukin 1β (IL-1β) etc, leading to hypersensitivity and exacerbating pain symptoms [9,10].
THP is the main bioactive components of the Chinese herbal medicine Corydalis Yanhusuo, which has been widely used for treating pain and cardiovascular disease in traditional Chinese medicine. Clinical and basic science researches have proved that it has good therapeutic effect in alleviating pain [11][12][13].
Studies have found that THP can speci cally block the activation of the p38-MAPK signaling pathway, and thus inhibit the in ammatory response in human monocyte cells [14]. So, can THP alleviate the pain symptoms of DNP rats, and what is the possible mechanism of this effect? Unfortunately, we have not found relevant literature reports. Therefore, we established a DNP rat model to observe the changes in pain threshold, the changes of microglia in the spinal cord, and the p38-MAPK-mediated in ammatory response to explore the effect of THP on DNP and its possible mechanism.

Preparation of DNP rats model and drug treatments
All the rats were randomly assigned to four groups : Blank control group (Blank) DNP model group (Model) THP therapy group (THP) and Methylcobalamin positive control group (MeCbl), n=15 per group. The model was established by high-fat and high-sugar diet combined with single dose intraperitoneal injection of STZ. In detail, rats of Model, THP and MeCbl groups were fed with high-fat and high-sugar (per 100g feed: ordinary feed 74.5g, lard 10g, sucrose 10g, egg yolk powder 5g, cholesterol 0.5g) while blank control group to be fed regularly. After 4 weeks, except the blank control group rats were injected with citric acid solution intraperitoneally, the other rats were injected with STZ solution 35mg/kg. After 72 hours, blood was collected from the tail vein to measure fasting blood glucose (FBG) and fasting insulin (FINS). And then, calculate the fasting insulin sensitivity index (ISI), ISI = In (FINS × FBG) -1 . A decreased in ISI as well as FBG ≥ 11.1 mmol•L -1 , were identi ed as a T2DM model rats. 2 weeks later, the MWT and TWL of T2DM rats were detected, and both of them fell below the baseline value of 85% were judged as DNP rats model. And then, drug intervention was given for 6 weeks, THP group rats were treated with THP by gavage according to their weight (4mg kg -1 ), MeCbl group rats were treated with mecobalamin (0.175mg kg -1 ), while distilled water (10 mL kg -1 ) for the Blank and Model groups (Fig. 2).

Determination of rat weight and FBG
The weight and FBG of rats in each group were measured before treatment and 2, 4 and 6 weeks after treatment (i.e. 0W 2W 4W 6W). After 12h of fasting with normal water supply, the rats were weighed and recorded. The tails of the rats were disinfected with alcohol cotton balls, and the FBG of rat tail venous blood was measured by glucose meter (type 5D-2, Yicheng Bioelectronics Technology, Beijing, China.).

Mechanical withdrawal threshold (MWT) test
We placed the rat on the cribriform metal plate , and separated it with a plexiglass cover. Using Von Frey laments (North coast, USA) with a test range of 0.008g-300g to vertically stimulate the right hind paw of the rats after the rat adapts for 30 minutes, and determine MWT by sequentially increasing and decreasing the intensity of the stimulus. When the laments are bent, rats aviod them by raising their legs or licking their feet are considered positive reactions. The measurement was performed 5 times in a row with an interval of 15s between each measurement. The mean of the minimum grams of positive reaction was regarded as the MWT of the rat.

Thermal withdrawal latency (TWL) test
The rats were put into the intelligent hot plate apparatus (type RB200, Taimeng Software, Chengdu , China) preheated to 55℃, and the timing was carried out at the same time. When the rat is heated and lick its hind feet, the time ends, and the contact time between the rat's hind feet and the hot plate is recorded. And the measurement was repeated three times, each rat was tested once every 15 minutes.
The average of the three results was calculated and recorded as the rat's TWL. It's important to note that the whole test process was kept quiet, and cleaning up the possible dirt in the cage in time after the rat is taken out to avoid affecting the accuracy of the next test results . The following thermocycling conditions were used: Initial denaturation at 95 °C And the relative mRNA expression levels were quantifed using the 2-ΔΔCq method. Primers for p38, OX42, β-actin were obtained from Tianyihuiyuan (Wuhan, China). The sequences were following: p38, forward, 5′-AGCAACCTCGCTGTGAATG-3′ , reverse, 5′ -ACAACGTTCTTCCGGTCAAC-3′; OX42, forward, 5′-CAAGGAGTGTGTTTGCGTGTC-3′ , OX42, reverse, 5′-TGAGTATGCCGTTCTTTGTTTC-3′; β-actin, forward, 5′

ELISA analysis
The blood was put into the non-anticoagulation tube, quiescenced in room temperature for 2 hours, and taking the supernatant after centrifugation at 3000 rpm for 15 minutes.

Statistical methods
GraphPad Prism 8.01 (GraphPad software, USA) was used to analyze the relevant data. The results are expressed in the form of mean ± standard deviation . All experiments were carried out at least three times. When comparing between groups, if the data satis es the normal distribution, choosing the oneway analysis of variance (ANOVA). The Kruskal-Wallis rank sum test is used if the data is not satis ed. Multiple comparisons adopting the Fisher's least signi cant difference (LSD). P< 0.05 was considered to be statistically signi cant, P< 0.05 was considered to be signi cant statistical signi cance.

THP ameliorated the weight loss and FINS rise in DNP rats but did not affect FBG and GSP
Results show that compared with the blank control group, the weight of rats in the remaining groups was signi cantly higher at 0W and there was no signi cant difference between those three groups. After 2 weeks of with or without treatment, the weight of rats in those three groups dropped to the lowest level and then gradually increased. Compareing with the model group, the weight of rats in the THP therapy group increased signi cantly at 4 and 6 weeks of treatment (Fig. 3A). Which indicates that THP can slow down the weight loss of rats due to DNP. In addition, comparing with the blank group, the serum levels of FINS in the model group increased signi cantly, after 6 weeks of treatment, the serum levels of FINS in the therapy group decreased signi cantly (Fig. 3B), indicating that THP could slow down the rise of FINS in DNP rat. However, THP had no signi cant effect on FBG and GSP in rats with DNP (Fig. 3C, D)

THP ameliorated pain symptoms in DNP rats
The results show that there were no signi cant changes in MWT or TWL of the blank group at each time point. But, the MWT and TWL values in the other groups were signi cantly decreased before treatment (0W) compared with the blank group, and there was no signi cant difference among those three groups. Meanwhile, the MWT and TWL of the THP therapy group and metacobalamin positive group signi cantly increased at 2W and 6W, which were signi cantly higher than those in the model group, indicating that THP had obvious analgesic effect on DNP rats (Fig. 4).

THP suppressed in ammation in DNP rats
The expression levels of IL-1β, TNF-α, IL6, CCL2, CCR2, CXCL1 and CXCR2 in the spinal cord of rats were detected by ELISA. Comparing with the blank group, the model group were signi cantly increased.
However, the expression levels of these factors were signi cantly lower after THP or methylcobalamin treatment than in the model group( g. 5) . And these results suggest that THP suppresses the in ammatory response in DNP rats.

THP inhibited the activation of microglia and p38-MAPK in the spinal cord of DNP rats
The expressions of p38, p-p38 and OX42 were signi cantly changed in both the model group and the therapy group. Western blotting assay results showed that the expression levels of p38, p-p38 and OX42 in the spinal cord of rats in the model group were signi cantly higher than those in the blank group ( Fig. 6A-C). And qRT-PCR assay shown the similar results (Fig. 6D, E). These results suggested that THP could inhibit the activation of microglia and p38-MAPK in the spinal cord of DNP rats.

THP inhibited the activation of microglia and p38-MAPK in the spinal cord horn of DNP rats
The expression of OX42 and p-p38 in the spinal cord horn of DNP rats and their co-labeling were observed by double-labelled immuno uorescence. After 6 weeks of treatment, the OX42 (red) and p-p38 (green) staining of the spinal cord horn was signi cantly increased in the model group compared with the blank group, and the number of co-labeled in microglia was increased too. Comparing with the model group, Ox42 and p-p38 staining as well as the number of co-labeled microglia of the spinal cord horn were decreased in the therapy group after treatment with THP. ( g. 7) These results suggest that THP induces a decrease in p-p38 expression and inhibit microglial activation in spinal cord horn of DNP rats.

Discussion
DNP is a painful and incurable complication in diabetic patients, characterized by increased sensitivity to mechanical and thermal stimuli [15]. We adopted high-fat and high-sugar feeding for 2 months in this study, and then a single intraperitoneal injection of STZ (35 mg/kg) was used to destroy the islets to simulate insulin resistance and establish a T2DM rats model [16]. After the T2DM model were established successfully, consistenting with the previous study, the MWT and TWL of the most of rats were signi cantly reduced within 2 weeks, which considered as successful DNP rats model [2,17].
THP is an active natural alkaloid isolated from Corydalis Yanhusuo which has used for treating pain and cardiovascular disease in traditional Chinese medicine [18,19] In neuropathic pain model mice, THP exerts obvious sedative, hypnotic and analgesic effects [20]. Similarly, THP showed an effective antihyperalgesic effect in a mouse model of neuropathic pain induced by oxaliplatin [21]. In the neuropathic pain model which was induced by segmental spinal nerve ligation, THP can relieve chronic in ammation and neuropathic pain in mice [22]. In this study, the weight of DNP rats increased signi cantly, and the MWT and TWL responses were improved to varying degrees, after 6 weeks of THP treatment. Based on these data, we speculate that THP may be an effective drug for the treatment of DNP, but the potential mechanism of THP remains to be further studied.
CXCL1 is a chemokine that promote both nociceptor and central sensitization via its main receptor CXCR2, which is a promising target for novel analgesic drugs [23].Studies have shown that CC motif chemokine ligand 2 (CCL2) and its receptor CCR2 play a key role in the occurrence and maintenance of neuropathic pain [24]. Intrathecal injection of CCL2 can produce obvious nociceptive and non-in amed pain behaviors in rats, and this situation can be reversed by intrathecal injection of CCR2 antagonists. Pre-injection of CCR2 antagonist into the intrathecal not only can prevent and relieve nerve pain but also can inhibit the activation of microglia and p38-MAPK [25][26][27] In addition, studies have shown that in neuropathic pain caused by CCI surgery, the expression of CXC motif chemokine ligand 1 (CXCL1) and its receptor CXCR2 in spinal dorsal horn neurons and DRG are increased, taking CXCR2 antagonists can reverse mechanical allodynia [28]. Further studies have found that CXCR2 and the activation of microglia may be involved in neuropathic pain, and p-p38 and p38-MAPK may be involved in it [29]. We also found that the levels of CCL2, CCR2, CXCL1 and CXCR2 in the spinal cord tissue of the model group increased in this study, and their expression decreased signi cantly after THP treatment. These results indicate that THP may alleviate the pain symptoms of DNP rats by regulating the corresponding chemokines and their receptors.
As a kind of resident nervous system phagocytes, microglia play an extremely important role in the occurrence and development of DNP along with its activation and corresponding pro-in ammatory reactions [30]. Studies have shown that in diabetic rats, microglia in the spinal cord are activated very early and are positively correlated with pain [31]. The increase expression of OX42 in the model group in this study also proved this, and the decreased of OX42 in the therapy group means that the activation of microglia has been inhibited by THP treatment. The persistent hyperglycemia state of diabetes leads to the activation of microglia, the phosphorylation of p38-MAPK and the release of pro-in ammatory factors (including IL-1β, IL-6 and TNF-α) [32].In addition, after peripheral nerve damage, spinal cord microglia are activated, leading to the secretion of in ammatory factors. In turn, in ammation can also activate p38-MAPK, thereby promote the activation of spinal cord microglia. In animal models of neuropathic pain, in ammatory factors are considered as a key factor in the occurrence and maintenance of hyperalgesia [33]. In this study, consistent with the reported results, we found that the protein levels of p38 and p-p38 in the spinal cord tissue of the model group increased signi cantly, while the expression of IL-1β, IL-6 and TNF-α increased. What's more, comparing with the model group, the relevant assays in the therapy group were signi cantly decreased after THP treatment, we speculate that this is the possible cause of THP alleviating the pain symptoms of DNP rats.
A widely accepted view is that OX42 is a cell surface marker for microglia activation, when peripheral nerves are injured, the morphology of microglia changes and the expression of microglia marker OX42 increases [33]. In the lipopolysaccharide (LPS) -stimulated BV2 microglia cells in ammation model and mouse in ammation model, p38-MAPK inhibitor have a signi cant inhibitory effect on both microglia activation and neuroin ammation [34]. And further research found that p38 activation in spinal microglia plays a key role in mechanical allodynia in rats [35]. In this study, We observed increased expression of the microglial cell marker OX42 in the spinal cord of DNP rats, accompanied by increased expression of p-p38, and an increased number of cells co-labeled with both. Fortunately, after THP treatment, their expression was signi cantly inhibited, which indicated that THP can reduce DNP by regulating the p38-MAPK signaling pathway in spinal microglia.
In summary, our research shows that DNP rats exhibit activation of microglia and activation of the p38-MAPK signaling in spinal cord. And we found that THP can improve the pain symptoms of DNP rats, and this effect may be achieved by reducing the in ammatory response and corresponding chemokines and their receptors. In addition, our research shows that THP can down-regulate the expression of OX42 and p-p38 in spinal microglia, thereby reduce the symptoms of DNP. In conclusion, our study revealed for the rst time that THP can exert a therapeutic effect on DNP rats by inhibiting the in ammatory response caused by microglia activation through the p38 pathway. It is bene cial to further develop the analgesic effect of THP for clinical application in DNP therapy Author Contributions: Aijuan Jiang designed the study. Lianzhi Cheng and Junlong Ma cooperated to complete experimental research and paper writing. Fanjing Wang analyzed the data. Kai Cheng, and Qian Chen modi ed the manuscript. All authors have seen and approved the manuscript and its contents. Figure 1 Chemical structure of THP. Molecular formula: C21H25NO4.