Anti-oxidative stress properties by Lactiplantibacillus plantarum SCS3 in streptozotocin-induced diabetic mice

ABSTRACT This study investigated the ability of Lactiplantibacillus plantarum SCS3 (L. plantarum SCS3) to alleviate oxidative stress in diabetic mice. Diabetic mouse model was established by administering 50 mg/kg (bodyweight) of streptozocin for 5 days. We then applied 0.5 mL of 1.0 × 1010 CFU/mL L. plantarum SCS3 cell suspension (TGa group), its intracellular contents (TGb group), or boiled intracellular contents (TGc group) to mice for ten weeks. Compared with the model group (MG), weight loss and hyperglycemia were most effectively ameliorated in the TGc group compared to the other treatment groups; based on the enzyme linked immunosorbent assay, levels of insulin and glycosylated hemoglobin were improved; the reactive oxygen species level decreased, while superoxide dismutase, catalase, and glutathione levels in the TGc mice increased significantly (P < .05). In conclusion, the boiled intracellular contents of L. plantarum SCS3 regulated oxidative stress and improved diabetes-related indicators in diabetic mice.


Introduction
Type 2 diabetes is characterized either by a relative insulin (INS) deficiency or INS resistance, thereby leading to elevated blood glucose levels (Luan et al., 2015). Oxidative stress is believed to play a key role in the nosogenesis of type-2 diabetes and its associated complications (Hamden et al., 2008). Oxidative stress contributes to the development of type 2 diabetes through three potential mechanisms: promoting the generation of reactive oxygen species (ROS), reducing the activity of antioxidant enzymes, and causing oxidative damage to deoxyribonucleic acid (DNA) (Bektas et al., 2018). In the ROS generation pathway, oxygen participates in high-energy electron transfer reactions to support the production of adenosine 5'-triphosphate (ATP) through oxidative phosphorylation (Burton & Jauniaux, 2011). This chemical reaction also produces free radicals, which damage nearby surrounding cells. Free radicals are biologically necessary to some extent because they play a role in stimulating repair despite simultaneously causing damage (Birben et al., 2012). However, an excess of free radicals can disrupt this repair process and cause oxidative stress. Free radical species include active oxygen and other molecules with unpaired electrons, which renders them unsteady and highly chemically reactive (Birben et al., 2012). To become more chemically stable, free radicals damage the molecules comprising DNA, proteins, and lipids, thereby resulting in significant tissue damage (Birben et al., 2012). Because of this, living systems are constantly being compromised by ROS. The human body contains various endogenous and exogenously-consumed antioxidants, including enzymatic antioxidants and non-enzymatic antioxidants (Birben et al., 2012). Because such a sophisticated antioxidant defense system exists within the body, these ROS attacks can be counter balanced. However, this balance is sometimes disturbed, leading to oxidative stress (Burton & Jauniaux, 2011).
In recent years, many researchers have demonstrated the antioxidant effects of probiotics by examining the antioxidant activity of lactic acid bacteria (Kim et al., 2005;Kullisaar et al., 2002). Lactiplantibacillus plantarum (L. plantarum) is a safe, food-grade microorganism with no significant side effects (Fan et al., 2012). It has also been shown to relieve lactose intolerance, treat diarrhea, rebalance host intestinal flora, inhibit the activity of intestinal cancer-promoting enzymes, regulate immunity, and exert other beneficial physiological effects (Wu et al., 2019). Studies have also demonstrated that some types of L. plantarum can improve the symptoms of type 2 diabetes in rats (Li et al., 2014(Li et al., , 2016. However, the mechanism by which L. plantarum improves type 2 diabetes is not yet clear. Furthermore, lactic acid bacteria and their active substances, such as protease and ferulic acid, may also regulate oxidative stress (Chen et al., 2012), but the key substances employed by L. plantarum are not yet defined. In the current study, we assessed the effects of three components obtained from L. plantarum SCS3 (its bacterial suspension, intracellular contents, and boiled intracellular contents) on the levels of blood glucose, insulin (INS), and glycosylated hemoglobin (HbA1c) in type 2 diabetic mice and investigated their effects on oxidative stress to clarify the mechanism and key substances of L. plantarum for improving type 2 diabetes. The obtained results provide a theoretical foundation for using L. plantarum SCS3 to prevent type 2 diabetes.

Preparation of different test substances
The bacterial strains were thawed and inoculated into MRS broth liquid medium at 0.5% inoculum volume (v/v) and incubated in a constant temperature incubator at 37°C for 16 h. The fermentation broth was centrifuged for 5 min (8000 × g, 4°C), after which, the supernatant was removed and the precipitated bacteria were washed three times with 0.1 mol/L sterile phosphate-buffered saline (PBS) (Zhang et al., 2020). The resulting precipitated bacteria were then divided into three parts. The first would be administered to the L. plantarum SCS3 suspension group (TGa) at (1.0 × 10 10 CFU/mL). The second portion of the precipitated bacteria had cell lysate added to it, after which it was shaken and mixed well. These cells were then lysed in an ice bath for 1 hour and centrifuged for 10 min (6000 × g, 4°C). Supernatant was subsequently obtained via centrifugation at 6000 × g for 10 min at 4°C and was then mixed with an equal volume of 0.1 mol/L sterile PBS. This resulting supernatant was then placed in an ice bath to be subsequently administered to the the L. plantarum SCS3 intracellular content group (TGb). The supernatant of the third portion of the precipitated bacteria was obtained similarly to the second portion of bacteria, but then placed into boiling water and heated for 30 min to ensure that the bacterial proteins were inactivated (suspension: PBS = 1: 15). The resulting would be subsequently administered to the the L. plantarum SCS3 boiled intracellular content group (TGc).

Animal experiments
The Sichuan Dashuo Experimental Animal Company (Chengdu, China) supplied 150 SPF Kunming male mice (3 weeks old, 36 ± 2 g). The study was authorized by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (2019-21) and conformed to the Directive (European Parliament, Council of the European Union, 2010). After a week of acclimation, the mice were randomly separated into 6 groups (25 mice/group), including the control group (KG), model group (MG), normal group (NG), TGa, TGb, and TGc. During the second week, all mice except for the NG mice received 50 mg/kg (bodyweight) of streptozocin (STZ) by intraperitoneal injection for 5 consecutive days to the diabetic mouse model (Lee et al., 2021). During this period, the mice were fed a standard diet every day. In the third week, the mice were fasted for 12 hours before their blood glucose levels were measured. The diabetic mouse model was considered to be successfully established when the fasting blood glucose (FBG) and 2 h postprandial blood glucose (PBG) levels of mice were more than 7.0 and 11.1 mmol/L, respectively (DeFronzo et al., 2015). After successful modeling and during the intragastric administration period, the NG and MG mice were fed normally, while the BG mice were administered 0.5 mL 0.1 mol/L PBS every day. Meanwhile, the TGa, TGb, and TGc mice were administered 0.5 mL of 1.0 × 10 10 CFU/mL L. plantarum SCS3 suspension, its intracellular contents, or its boiled intracellular contents, respectively, for ten weeks.

Determination of weight
The body weights of the mice were recorded weekly, each following a 12 h fast, until the 10th week.

Blood glucose measurements
Venous tail blood was collected following 12 h fast and analyzed for glucose with a blood glucose meter (Taiwan Qingli Biotechnology Co., Ltd., China) every week. The postprandial 2 h blood glucose levels of the mice were also measured once a week until the 10th week.

Oral glucose tolerance test
An oral glucose tolerance test (OGTT) was performed at the first and 10th week after successful modeling respectively. The mice were fasted for 12 h and supplemented with water. We measured their blood glucose levels at 0, 15, 30, 60, 90, and 120 min after the administration of 2.0 g/kg (bodyweight) of glucose directly into the stomachs of the mice (Huang et al., 2018).

Statistical analysis
Data were expressed as the mean ± standard deviation (× ± s). Statistical analysis was performed using GraphPad Prism for Mac OS, version 9.0.2 (GraphPad Software, San Diego, California, USA). Comparisons between each group and the normal group or comparisons between each treatment group and the model group were performed by One-way ANOVA followed by Dunnett's multiple comparisons test, and P < .05 was considered statistically significant.

Effect of L. plantarum SCS3 on the body weights in mice
The mice in all groups lost weight in the second week of successful modeling, as shown in Figure 1. After the eighth week, the weights of the TGa, TGb, and TGc mice began to increase. Moreover, at the end of administration (the tenth week), the weight loss trend in the TGa, TGb, and TGc mice was significantly improved compared with that of the MG mice (P < .05). Compared with TGa and TGb mice, the weights of TGc mice were the highest at the conclusion of the experiment. These results indicate that L.plantarum SCS3, especially its boiled intracellular content, significantly ameliorated the trend of weight loss observed in the TG3 mice (P < .05).

Effect of L. plantarum SCS3 on the blood glucose levels
The FBG levels in the mice increased from the third week ( Figure 2a) except for the NG mice. The highest measured FBG levels in the MG and BG mice were obtained in the eighth to ninth weeks. The FBG levels of the TGc mice decreased after the sixth week and remained stable for four weeks. Meanwhile, after mice were injected with STZ, the PBG levels of all mice except for those in the NG mice increased from the second week onwards (Figure 2b). Notably, the PBG of the TGc mice decreased from the fifth week and then maintained steady for five weeks. At the closing of the experiment, the TGc mice exhibited significantly decreased PBG levels compared with the MG mice (P < .05). These results suggest that the FBG and PBG levels were improved by the boiled intracellular contents of L. plantarum SCS3.

Effect of L. plantarum SCS3 on oral glucose tolerance in mice
Results of the OGTT conducted in the first and tenth weeks during the gavage treatment are shown in Figure 3. In the first week, the blood glucose levels for all groups increased after ingestion and reached their peak levels at 15 min. Compared with the NG mice, the blood glucose levels of the MG, BG, TGa, TGb, and TGc mice were higher, as shown in Figure 3a. Significant glucose AUC values for these groups were also observed, as shown in Figure 3b (P < .05), which suggests that the MG, BG, TGa, TGb, and TGc mice had impaired blood glucose clearance. After ten weeks of the gavage treatment, decreased blood glucose levels at 2 h occurred in the TGa, TGb, and TGc mice compared with the MG mice ( Figure 3c). We also found that compared  1. Efecto de L. plantarum SCS3 en el peso corporal de ratones. Sin tratamiento (NG). Tratamiento con STZ+PBS (BG). Tratamiento con STZ (MG). Tratamiento con STZ+suspensión de L. plantarum SCS3 (TGa). Tratamiento de STZ+contenido intracelular de L. plantarum SCS3 (TGb). Tratamiento de contenido intracelular STZ+L. plantarum SCS3 hervido (TGc). *Diferencia significativa (P<0.05) entre cada grupo de tratamiento y los ratones MG.  with the MG mice, the glucose AUC values for the TGa, TGb, and TGc groups reduced significantly to 2776, 2773, and 2899, respectively (P < .05) (Figure 3d). These results indicate that L. plantarum SCS3 improved the oral glucose tolerance of the treated mice compared with the MG group.

Effect of L. plantarum SCS3 on INS and HbA1c in mice
As shown in Figure 4a, the INS levels increased in the BG and MG mice. This indicated that more INS was produced in order to counteract the hyperglycemia experienced by the diabetic mice. After ten weeks of gavage treatment, the TGb and TGc groups exhibited lower INS levels relative to the MG group, and the INS level of the TGc group in particular decreased. There was no significant difference observed in the NG group (P > .05). Meanwhile, the HbA1c levels of TGa, TGb, and TGc mice were also significantly decreased (P < .05) compared with that of the MG mice, as shown in Figure 4b. These results reveal that the levels of INS and HbA1c in the TGc mice were the lowest among the three test groups.

Effect of L. plantarum SCS3 on ROS and MDA levels in mice
As shown in Figure 5a, the ROS levels of the TGa, TGb, and TGc mice were lower than that of the MG mice after ten weeks of administration, and the ROS level of the TGc mice decreased more remarkable among the three test groups. These results indicated that the oxidative damage of diabetic mice caused by ROS was alleviated by the boiled intracellular contents of L. plantarum SCS3. However, the TGc mice exhibited a higher MDA level compared with the MG mice, as shown in Figure 5b.

Effect of L. plantarum SCS3 on antioxidant levels in mice
We observed that, except for HO-1, the levels of other antioxidants were decreased in the MG and BG mice, suggesting that the antioxidants were negatively affected in the diabetic mice. After ten weeks of gavage treatment, the levels of SOD, GPx, CAT, NQO1, γ-GCS and GSH in the TGa, TGb, and TGc mice were increased, while the level of HO-1 had not increased in TGb and TGc mice, the levels of GST in TGb mice had not increased. Worth noting is that the levels of SOD, CAT, NQO1, γ-GCS and GSH were remarkably different between the MG and TGc mice (P < .05), as shown in Figure 6(a,c,e,f,h). These results suggest that the boiled intracellular contents of L. plantarum SCS3 showed the most potent effect out of the three treatments on antioxidants levels recovery in type 2 diabetic mice.  3. Efecto de L. plantarum SCS3 en la OGTT de ratones. Sin tratamiento (NG). Tratamiento STZ+PBS (BG). Tratamiento con STZ (MG). Tratamiento de STZ +suspensión de L. plantarum SCS3 (TGa). Tratamiento de STZ+contenido intracelular de L. plantarum SCS3 (TGb). Tratamiento de contenido intracelular STZ +L. plantarum SCS3 hervido (TGc). (a) Cambio en los niveles de glucosa de la sangre de ratones durante la primera semana; (b) cambio en el AUC de la glucosa en la sangre de ratones; (c) cambio en los niveles de glucosa de la sangre de ratones en la décima semana; (d) cambio en el segundo AUC de la glucosa en la sangre de ratones. *Diferencia significativa (P < .05) entre cada grupo de tratamiento y los ratones MG. #diferencia significativa (P < .05) con respecto a NG.

Discussion
This study revealed that weight loss and the FBG, PBG, and OGTT levels in type-2 diabetic mice could be improved by L. plantarum SCS3, especially its boiled intracellular contents. These results are consistent with those obtained by previous studies (Tabuchi et al., 2003;Yadav et al., 2018). Lactobacillus GG significantly improved glucose tolerance in a neonatal streptozotocin-diabetic rat model. Homoplastically, the feeding of probiotic fermented milk containing Lactobacillus rhamnosus MTCC: 5957, Lactobacillus rhamnosus MTCC: 5897 and Lactobacillus fermentum MTCC: 5898 significantly improved fasting blood glucose levels. Regarding INS, it is suspected to function as a key hormone in the growth and development of tissues and the control of glucose homeostasis (Rains & Jain, 2011). Chronically high levels of INS can result in INS resistance, which is a major cause of type 2 diabetes (Rains & Jain, 2011). Moreover, the indicator HbA1c reflects the average blood glucose concentration over the previous few months (Yoshii et al., 2022). The present study showed that the boiled intracellular contents of L. plantarum SCS3 also decreased the INS and HbA1c levels. Similarly, fermented Momordica charantia L. with Lactobacillus plantarum NCU116 could reduce serum insulin and HbA1c levels in diabetic rats (Gao et al., 2018). In contrast, the contents of INS in mice administered L. plantarum SCS2 were significantly increased compared to those in MG mice, as shown in our previous work (Wu et al., 2021). This may be attributable to the different stages of diabetes to which the mice are subjected to.
were found to be consistent with the results of the study. Some recent studies have demonstrated that varieties of L. plantarum can decrease the levels of MDA levels (Lin et al., 2018;Zhang et al., 2017). However, the MDA level measured in the TGc group in our study was increased compared to the MG group, thereby showing our research results to be inconsistent with those of previous studies. We therefore speculate that the boiled intracellular contents of L. plantarum SCS3 lacks certain substances, such that it can only prevent the formation of new free radicals, but not extinguish the reactivity of preexisting free radicals. The resulting metabolites produced by these free radicals may then fail to undergo excretion from the body. ROS are more unstable than MDA and have a shorter retention time in the body (Cherian et al., 2019). Therefore, MDA that has been originally produced in the body is more likely to accumulate compared to ROS, and one consequence of this could be that the ROS level of the TGc mice decreased while the MDA level increased.
Our results showed that the TGa, TGb, and TGc groups exhibited effective recoveries of SOD, GPx, CAT, NQO1, γ-GCS and GSH levels. In particular, the antioxidants SOD, CAT, GSH, and GPx each perform a vital role in counteracting oxidative stress, and so most of the literature has focused on these antioxidants. Li et al. (2014) and Li et al. (2016) demonstrated that L. plantarum NCU116 and L. plantarum CCFM0236 could both significantly increase the activities of SOD, CAT, and GPx. However, Valentini et al. (2015) reported results showing that L. plantarum did not significantly increase GPx levels. These potentially conflicting results may have been due to differences in nutritional, genetic, and environmental factors that affect the plasma redox state. Of note, the activity of some antioxidant enzymes is influenced by genetic polymorphisms (Arpaci et al., 2020;Jiménez-Osorio et al., 2014). Furthermore, the current study found that the boiled intracellular contents of L. plantarum SCS3 did not substantially affect the level of HO-1 in the type 2 diabetic mice, which may be attributable to the fact that HO-1 production is controlled by other transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) (Negi et al., 2015).
Overall, the TGc mice exhibited the lowest FBG, PBG, and OGTT levels while having the highest weight among the three treatment groups. The TGc treatment also had a potent effect on the recovery of the levels of INS and HbA1c at ten weeks of administration. These results indicated that the boiled intracellular contents of L. plantarum SCS3 can ameliorate weight loss and reduce blood glucose, INS, and HbA1c levels. Furthermore, levels of the key enzymes SOD and GPx in the TGc mice were the highest among the three treatment groups. Tang et al. (2017) found that the intact cell suspension of L. plantarum MA2 had the strongest antioxidant capacity, which may be related to the attribution of some cell surface-active compounds such as proteins, polysaccharides, and lipoteichoic acid. Meanwhile, the TGc mice had the lowest ROS levels of the three treatment groups. The ROS produced in any given cell relies on endogenous antioxidant enzymes to maintain homeostasis, whereby the levels of antioxidant enzymes should increase to manage oxidative stress (Ahmed et al., 2006). Moreover, Tangvarasittichai (2015) confirmed that an increase in oxidative stress could lead to INS resistance, pancreatic β-cell dysfunction, and impaired glucose tolerance, thereby promoting the occurrence and progression of type 2 diabetes. In summation, we speculate that the mechanism by which L. plantarum SCS3 improves diabetes is that its' boiled intracellular contents can enhance the levels of the important antioxidants, as observed in the TGc group, and thereby effectively scavenge ROS, ameliorate oxidative stress to a certain extent, ameliorate INS resistance, improve INS and HbA1c levels, reduce the blood glucose levels, and facilitate the regaining or maintenance of bodyweight. However, this study did not investigate the effect of the boiled intracellular content of L. plantarum SCS3 on the oxidative stress in the pancreases of diabetic mice, and it is not clear whether there exists a direct correlation between the improvement of diabetes-related markers and the reduction of oxidative stress in the model mice used in the study. In the next stage of this research, we will focus on the key substances and biological characteristics of the boiled intracellular contents of L. plantarum SCS3 that improve oxidative stress and blood glucose levels in type-2 diabetic mice, and investigate the underlying mechanism for improving diabetes-related symptoms by oxidative stress regulation.

Conclusion
Results showed that the boiled intracellular contents of L. plantarum SCS3 could alleviate oxidative stress and diabetes-related indicators in diabetic model mice. The possible mechanism underlying these results may be that the boiled intracellular contents of precipitated bacteria maintain pancreatic β-cell homeostasis by relieving the oxidative stress response, thereby reducing blood glucose levels. The specific substances that play a role in L. plantarum SCS3 are likely to specifically reside within the boiled intracellular contents.