Abstract
The current study aims to investigate the effect of rhein lysinate (RHL) on neuromotor function in rats with spinal cord injury (SCI) and to explore the underlying mechanism. A total of 63 adult male SD rats were randomly divided into the sham group, SCI group and RHL group (SCI‐RHL group), with 21 rats in each group. Basso Beattie Bresnahan (BBB) scoring and the inclined plate test were used to evaluate the changes in motor function after SCI. Then, superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), and malondialdehyde (MDA) contents were quantified. Caspase‐3‐positive cells in spinal cord tissue were detected by immunohistochemical (IHC) staining, and protein expression was detected by Western blots. Here, our data showed that compared with the SCI group, the BBB score and inclined plate test score of the SCI‐RHL group were significantly higher than those of the SCI group 7 days after the RHL intervention. After 7 days of RHL treatment, the activities of SOD and GSH‐Px in the spinal cord increased significantly. At the same time, RHL reduced the MDA content in spinal cord tissue of SCI rats. Moreover, cleaved‐caspase‐3‐positive cells and apoptotic cells were significantly lower in the SCI‐RHL group than in the SCI group. More importantly, p38 mitogen‐activated protein kinase (p38 MAPK) was significantly decreased in the SCI‐RHL group compared with the SCI group. In summary, RHL can inhibit the activation of the p38 MAPK pathway after SCI, thereby reducing the apoptosis of spinal cord neurons and improving the neuromotor function of SCI rats.
Keywords: neuronal apoptosis, p38 mitogen‐activated protein kinase, rhein lysinate, spinal cord injury
1. INTRODUCTION
Spinal cord injury (SCI) is a serious threat to human health, which includes primary and secondary injuries.1, 2 Neuronal apoptosis is an important component of secondary SCI and spinal nerve cell death after SCI is caused by apoptosis.3, 4, 5 Studies in different SCI models have confirmed that neuronal apoptosis is widespread in secondary SCI.6, 7 Apoptosis, also known as programmed cell death, is an intrinsic natural physiological process that maintains the stability of various tissues in the human body by eliminating metabolites of dead cells.7 The mechanism of apoptosis in nerve cells is very complicated.8 It is suggested that various posttraumatic stimulation signals can induce apoptosis through different pathways.1, 6
Mitogen‐activated protein kinase (MAPK) is a kind of serine/threonine protein kinase that is important signal transducer of cell apoptosis.9, 10 After extracellular stimulation, such as cytokines and neurotransmitters, the signals can be transferred to the nucleus for gene expression and regulation.11 The three most important MAPK signaling pathways are the extracellular regulated protein kinases (ERK) pathway, the p38 pathway, and the c‐jun N‐terminal kinase (JNK) pathway.12 The p38 MAPK pathway is considered to be a classical MAPK pathway and plays an important role in the apoptosis network caused by secondary SCI.13 Studies have shown that the p38 MAPK signaling pathway is activated after SCI, and the activated p38 MAPK signaling pathway can further aggravate the injury, so inhibition of the p38 MAPK signaling pathway after SCI may be an effective treatment for secondary SCI.9, 14
Rhein (RH) is one of the main active ingredients of traditional Chinese medicine, such as rhubarb and Polygonum multiflorum.15 Rhein lysinate (RHL) was obtained by structural modification of RH with lysine (LY).16 Its water solubility was significantly improved, and its clinical application was expanded.17 Studies have shown that RHL is characterized by anti‐inflammatory and antitumor features.17, 18 In human glioma U87 cells, RHL is shown to induce cell apoptosis via suppressing Bcl‐2 and increasing Bax expression.19 In cervical cancer cells, RHL is found to suppress HeLa cell growth via p38 MAPK and JNK signaling pathway.20 However, whether RHL improves SCI and the underlying mechanism has not been reported.
2. MATERIALS AND METHODS
2.1. Experimental animals
Sixty‐three healthy adult male SD rats, weighing 250 to 300 g, were provided by the Animal Experimental Center of the Chinese Academy of Sciences (Shanghai, China). According to the random number table method, they were divided into a sham group, SCI group and RHL treatment group, with 21 rats in each group. Rhein (Nanjing Qingze Pharmaceutical Science and Technology Development Co., Ltd., Cat no: 080826, purity: 98%) and L‐lysine (Beijing Science and Navy Boat Biotechnology Development Center, Cat no: 081007, purity: 98%) were obtained. The synthetic method of RHL is as described above.21
SD rats in the SCI group and the RHL treatment (SCI‐RHL) group were anesthetized by intraperitoneal injection of 10% chloral hydrate (4 mL/kg) after adaptive feeding for 1 week. In the SCI group, skin was routinely disinfected, and a longitudinal incision was made on the dorsal side of the spine to expose the spinous processes and laminae. The tenth thoracic spine (T10) spinous process and laminae were cut, and the spinal cord was exposed to endure 10 g of sterilized metal rods from 25 mm. Successful SCI occurs when congestion and transient spastic tail swing appear. The rats were treated for 4 weeks. The RHL group was given RHL solution at a dose of 70 mg/kg.d at the same time after SCI, while the control group and the model group were given normal saline. During the entire experiment, rats were given free access to food and water.
For the sham group, anesthesia and fixation methods were the same as in the SCI group, but only T10 laminotomy was performed without injury to the spinal cord. All experiments were conducted in accordance with the relevant regulations of the Laboratory Animal Ethics Committee of Shenzhen Pingle Orthopedic Hospital.
2.2. Sampling and section preparation
Seven days after modeling, six rats in each group were randomly selected. After anesthesia with chloral hydrate, 0.1 M PBS pH 7.4 buffer solution was perfused into the heart. After perfusion with 4% polyformaldehyde, the organs were fixed. The injured spinal cord was dissected for 20 mm, immersed in 4% polyformaldehyde overnight, and then placed in 30% sucrose solution for determination. Fixed tissues were dehydrated routinely, paraffin embedded, and sliced vertically or horizontally with a thickness of 10 μm.
2.3. Motor function assessment
Five rats were taken from each group. To evaluate the changes in motor function after SCI, the motor function of the hind limbs of rats was evaluated by the BBB score and the inclined plate test before and after operation on the 1st, 3rd, 7th, 14th, 21st, and 28th days. BBB Score: The motor function of the hind limbs of rats was divided into 22 grades; the total paralysis score was 0, and the normal score was 21. The score was determined at 9 pm by a double blind method. Each rat was measured three times, and the average value was taken. The inclined plate test: the rats were placed on a special inclined plate with shallow grooves on the test face, and the ability to maintain posture, grasping ability and holding ability on the inclined plate were measured with a maximum angle for 5 seconds. Each rat was assessed three times, and the highest score was recorded. The maximum angle of normal rats on the inclined plate was 80°. Relevant scores were determined by the double‐blind method.
2.4. Detection of the apoptotic level of spinal cord neurons
According to the TUNEL Apoptosis Assay Kit, apoptosis of spinal cord neurons was detected in rats after administration of RHL or saline for 4 weeks. The tissue sections were washed with PBS three times and incubated with 20 μg/mL proteinase K at room temperature for 15 minutes. The sections were washed twice with PBS, then incubated with 37°C in TUNEL reaction mixture for 60 minutes and washed with PBS three times for 3 minutes. The sections were incubated at room temperature for 30 minutes with HRP‐streptomyces antibiotic protein reagent diluted with PBS (1:200). Optical microscopy was used to collect pictures. Five visual fields were randomly selected for each tissue slice to observe the number of apoptotic bone marrow cells in each group.
2.5. Detection of MDA content and SOD and GSH‐Px activity in spinal cord tissue
MDA content was determined by the thiobarbituric acid method, SOD activity by the biphenyltriphenol autoxidation method, and GSH‐Px activity by the NADPH coupling method, and the operation was strictly in accordance with the kit instructions (Cat: 003‐1, A001‐3, A005, Nanjing Jiancheng Bioengineering Institute, Nanjing City, China). All of the quantification was completed in rats after administration of RHL or saline for 4 weeks.
2.6. Detection of Caspase‐3‐positive cells in spinal cord tissue
Fixed spinal cord tissues were collected in rats after administration of RHL or saline for 4 weeks. They were then dehydrated, made transparent and embedded, made into 5 μm paraffin sections, baked for 2 hours, dewaxed, hydrated with gradient alcohol, repaired with high‐pressure antigen, and cooled naturally. Immunohistochemistry (IHC) SP was used to remove endogenous peroxidase activity by incubation with 3% H2O2 and then washed several times in 0.1 M PBS buffer. At room temperature, the sections were incubated in 0.15% polyethylene glycol octyl phenyl ether and sealed with 1% goat serum for 1 hour. Next, the sections were incubated overnight with rabbit anti‐Caspase‐3 antibody (1:200, CST). Then, the sections were incubated with horseradish peroxidase for 2 hours. Finally, the sections were dyed, dehydrated, made transparent, sealed, and dried. In the negative control, PBS was used instead of primary antibody, and the other steps were the same. Finally, five visual fields were randomly selected from each slice by optical microscopy, and the number of Caspase‐3‐positive cells was calculated using the ImageJ software.
2.7. Western blot analysis
Three rats in each group were randomly selected at 8:00 to 10:00 am on the 1st, 3rd, 5th, 7th, and 28th day after operation. The injured spinal cord was dissected 20 mm from the injured site. The injured spinal cord was quickly injected into liquid nitrogen and stored at −80°C for reserve. The levels of p‐ERK, p‐p38, ERK, and p38 were measured by Western blot analyses. The expression levels of caspase‐3, caspase‐9, polyadenosine diphosphate‐ribose polymerase (PARP), and lymphocytoma‐2 gene (Bcl‐2) in spinal cord injured 7 days after operation were studied. The expression of β‐actin was used as an internal reference. The extract of 12% SDS‐PAGE protein isolate was 50 μg and transferred to a PVDF membrane. At room temperature, 5% skim milk powder was dissolved in TBST containing 0.5% Tween 20 and sealed for 1 hour. The PVDF membrane was incubated overnight with 4°C in diluted primary antibody. TBST was used for three washes, and the membrane was incubated with horseradish peroxidase‐labeled antibody for 2 hours at room temperature. Enhanced chemiluminescence (ECL) was used, and the protein expression was analyzed by the ImageJ software.
2.8. Statistical analyses
The data are represented as the mean ± SD (SD). Two‐tailed unpaired Student t tests were used for comparisons of two groups. The one‐way ANOVA multiple comparison test (SPSS 13.0) followed by a Turkey post hoc test was used for comparisons of more than two groups. P < .05 was considered statistically significant.
3. RESULTS
3.1. Motor function evaluation in each group
The BBB score and inclined plate test score in the SCI group were significantly lower than those in the Sham group. Here our data showed that RHL did not improve the motor function within 1 week after SCI. In contrast, RHL only slightly but significantly increases the BBB and incline plate test scores after 2 weeks of SCI, indicating that RHL could improve motor function after SCI (Figure 1A,B).
Figure 1.
Motor function was evaluated using the BBB score and inclined plate test score in each group. A, The BBB score and B, inclined plate test score in the SCI group were significantly lower than those in the sham group, while the SCI‐RHL group was significantly higher than those in the SCI group. (n = 5 for each group) *P < .05, ***P < .001 vs sham group; # P < .05 vs SCI group
3.2. RHL improved antioxidant activity in spinal cord tissues after SCI
Next, we evaluated the effect of RHL on oxidative stress after SCI. Compared with the sham group, the SOD and GSH‐Px activities in spinal cord tissue of SCI rats decreased significantly, while the MDA content increased significantly. After 7 days of RHL treatment, the SOD and GSH‐Px activities in the spinal cord increased significantly. At the same time, RHL also reduced the content of MDA in spinal cord tissue of SCI rats, with statistical significance (Figure 2A‐C). Meanwhile, TUNEL staining demonstrated that apoptotic cells were significantly higher in SCI rats than in the sham rats. However, treatment with RHL after 7 days significantly decreased apoptosis in spinal cord tissues post SCI (Figure 2D).
Figure 2.
RHL improved antioxidant activity in spinal cord tissues after SCI. The levels of SOD (A), GSH‐Px (B), and MDA (C) were quantified in the spinal cord tissues of the three groups. (n = 5 for each group) (D) TUNEL staining demonstrated that treatment with RHL after 7 days significantly decreased apoptosis in spinal cord tissues post SCI (scale bar represents 50 μm). (n = 5 for each group). *P < .05, ***P < .001 vs sham; # P < .05 vs SCI
3.3. RHL reduced the expression of cleaved‐caspase3 in spinal cord tissues after SCI
Caspase‐3, caspase‐9, PARP, and Bcl‐2 are widely involved in SCI‐related cell apoptosis.22 Hence, we explored the effect of RHL on their expression. As shown in Figure 3A, after SCI, the expression of proapoptotic proteins, including cleaved caspase‐3, cleaved caspase‐9, and cleaved PARP, was significantly increased, while the expression of the antiapoptotic protein Bcl‐2 was significantly decreased. In contrast, treatment with RHL after 7 days significantly reversed these changes (Figure 3A). IHC staining also demonstrated that c‐Caspase3‐positive cells were significantly increased in SCI rats but decreased after RHL treatment (Figure 3B).
Figure 3.
RHL reduced the expression of apoptosis‐related proteins in spinal cord tissues after SCI. A, Western blot assays demonstrated that treatment with RHL after 7 days significantly decreased the expression of cleaved caspase‐3, cleaved caspase‐9 and cleaved PARP but enhanced the expression of Bcl‐2. B, IHC staining also demonstrated that c‐Caspase3‐positive cells were significantly increased in SCI rats but decreased after RHL treatment. (n = 3 for each group) *P < .05, **P < .01, ***P < .001 vs sham; #P < .05, ## P < .01 vs SCI
3.4. RHL inhibited the activation of p38 MAPK signaling after SCI
ERK1/2, JNK1/2 and p38 MAPK are three important members of the MAPK family, which play key roles in cell apoptosis after SCI.23 Hence, we explored the effect of RHL on their activation. As shown in Figure 4, after SCI, ERK1/2 signaling was activated compared with that of the sham group. However, no significant effect of RHL was observed on the inactivation of ERK signaling after SCI for 3 days, 7 days, 14 days and 21 days (Figure 4). In addition, slight increase of p‐JNK1/2 was observed in the spinal cord tissues of SCI rats compared with that of sham. However, RHL was not found to inhibit the phosphorylation levels of JNK1/2 in SCI rats (Figure 4). In comparison, activation of p38 MAPK signaling was obvious after SCI compared to that of the sham group (Figure 4). More importantly, treatment with RHL reduced the phosphorylation levels of p38 MAPK compared with the SCI group after SCI for 3 days, 7 days, and 14 days, but the inhibitory effect of RHL on p38 phosphorylation was not observed at 21d (Figure 4). These data indicated that RHL is an inhibitor of p38 MAPK activation after SCI.
Figure 4.
RHL inhibited the activation of p38 MAPK signaling after SCI for 3 days, 7 days and 14 days. (n = 3 for each group) **P < .01 vs sham; # P < .05 vs SCI
3.5. Discussion
Approximately 500 000 people suffer from SCI worldwide every year.24 Most SCI patients suffer from paraplegia and bed rest for a long time, which results in a series of complications, such as deep vein thrombosis and bedsores of the lower limbs, and brings heavy burden to patients and their families.25 The recovery of motor function is often determined by nerve repair after SCI.26 Rhubarb has effects against cerebral ischemia injury, can alleviate brain tissue injury after ischemia‐reperfusion, and has a protective effect on nervous system injury caused by cerebral ischemia‐reperfusion.27, 28 Rhein is one of the effective components of rhubarb, and its protective effects on cerebrovascular conditions and tumors have been reported.29, 30 RHL is a new compound produced by the reaction of lysine and rhein, which can dissolve in water and is convenient for drug administration.18 In this study, for the first time, we explored the effects of RHL on SCI in rats and evaluated the specific molecular mechanisms.
Neuromotor function of rats is an important criterion for observing SCI and its recovery.31 Sensory and functional recovery is the most important and direct observation index for SCI treatment.8 BBB scores and inclined plate tests were used to assess the severity of SCI. The results showed that compared with the SCI group, the BBB score and inclined plate test score of the SCI‐RHL group increased significantly after 7 days of RHL intervention, with statistical significance. This finding suggests that RHL can promote the recovery of neuromotor function after SCI in rats. Here, RHL was not shown to improve the motor function within 1 week after SCI, but improved it after 2 weeks. These may due to the fact that RHL was administrated at the same time after SCI.
Apoptosis is a gene‐controlled cell suicide process through which nuclear metabolites of dead cells can be removed to maintain the stability of various systems in the human body.32 Apoptotic phenomena are widespread after SCI.1 Apoptosis of neurons and oligodendrocytes is a prominent feature of secondary degenerative response, which eventually leads to progressive degeneration of the spinal cord.32 Apoptosis after SCI is regulated by multiple genes, including Caspase‐3, Caspase‐9, PARP, and Bcl‐2.32, 33 Some studies have found that Caspase‐3 is the most important protease in the process of apoptosis, which not only participates in the initiation of apoptosis but also participates in the execution of the apoptotic process.34, 35 The results showed that the expression of Caspase‐3 in spinal cord tissue of the sham‐operated group was weak and that the number of apoptotic cells was small. In contrast, the expression of Caspase‐3 in spinal cord tissue increased significantly after SCI, and the number of apoptotic cells increased significantly. Compared with the SCI group, the number of Caspase‐3‐positive cells and apoptotic cells in the spinal cord tissue of the SCI‐RHL group decreased significantly 7 days after RHL intervention. These results suggest that RHL can inhibit the expression of Caspase‐3‐positive cells in the rat spinal cord and reduce the apoptosis of neurons after SCI.
Next, we evaluated the underlying mechanism by which RHL can improve functional recovery and promote neuroprotection after SCI. By affecting gene transcription and regulation in animal cells, the MAPK family influences cell proliferation, differentiation, transformation and apoptosis, including ERK1/2 MAPK family, the p38 MAPK family and JNK1/2.13, 36 In various cancer cells, activation of ERK is reported to enhance cell proliferation.37, 38 Here, our data showed that ERK phosphorylation was significantly enhanced after SCI, but RHL treatment could not suppress the activation of ERK. These findings indicate that ERK is not inhibited by RHL in spinal cord tissues after SCI. Previous study has also shown that JNK1/2 was involved in response to stress stimuli.36 Hence, we also evaluated the effects of RHL on JNK1/2 phosphorylation. Our data showed that RHL could not inhibit JNK activation. The results showed that RHL‐induced cell apoptosis was not mediated by the activation of JNK in spinal cord tissues after SCI. In various systems, the proapoptotic/growth inhibitory effects of p38 MAPK has been reported.39, 40 The results showed that the positive expression of p38 MAPK in the SCI group was significantly higher than that in the sham operation group, indicating growth inhibition in spinal cord tissues. Compared with the SCI group, the positive expression of p38 MAPK in the SCI‐RHL group decreased significantly after RHL treatment, and the difference was statistically significant. This finding suggests that RHL can inhibit the p38 MAPK pathway after SCI, thereby reducing the apoptosis of spinal cord neurons to protect nerve function.
In conclusion, RHL inhibits the activation of the p38 MAPK pathway after SCI, which can reduce the apoptosis of spinal cord neurons and improve neuromotor function in rats with SCI.
CONFLICT OF INTEREST
All authors declare no conflict of interest.
Hao J, Wang P, Pei D‐P, Jia B, Hu Q‐S. Rhein lysinate improves motor function in rats with spinal cord injury via inhibiting p38 MAPK pathway. Kaohsiung J Med Sci. 2019;35 765–771. 10.1002/kjm2.12123
Funding information Doctor initial fund from Shenzhen Pingle Orthopedic Hospital, Grant/Award Number: 20170648
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