Multi-strain probiotic supplement attenuates streptozotocin-induced type-2 diabetes by reducing inflammation and β-cell death in rats

Probiotics are health beneficial bacterial populations colonizing the human gut and skin. Probiotics are believed to be involved in immune system regulation, gut microbiota stabilization, prevention of infectious diseases, and adjustments of host metabolic activities. Probiotics such as Lactobacillus and Bifidobacterium affect glycemic levels, blood lipids, and protein metabolism. However, the interactions between probiotics and metabolic diseases as well as the underlying mechanisms remain unclear. We used streptozotocin (STZ)-induced diabetic animal models to study the effect of ProbiogluTM, a multi-strain probiotic supplement including Lactobaccilus salivarius subsp. salicinius AP-32, L. johnsonii MH-68, L. reuteri GL-104, and Bifidobacterium animalis subsp. lactis CP-9, on the regulation of physiochemical parameters related to type-2 diabetes. Experimental rats were randomly assigned into five groups, control group, streptozotocin (STZ)-treated rats (STZ group), STZ + 1× ProbiogluTM group, STZ + 5× ProbiogluTM group, and STZ + 10× ProbiogluTM group, and physiological data were measured at weeks 0, 2, 4, 6, and 8. Our results indicate that supplementation with ProbiogluTM significantly improved glucose tolerance, glycemic levels, insulin levels, and insulin resistance (HOMA-IR). Furthermore, we observed reduction in urea and blood lipid levels, including low-density lipoprotein (LDL), triglycerides (TG), and total cholesterol (TC). ProbiogluTM administration increased the β-cell mass in STZ-induced diabetic animal models, whereas it reduced the levels of proinflammatory cytokines TNF-α, IL-6, and IL-1β. In addition, the enhancement of oxidative stress biomarkers and superoxide dismutase (SOD) activities was associated with a decrease in malondialdehyde (MDA) levels. We conclude that ProbiogluTM attenuates STZ-induced type-2 diabetes by protecting β-cells, stabilizing glycemic levels, and reducing inflammation. Among all probiotic treating groups, the 10×ProbiogluTM treatment revealed the best results. However, these experimental results still need to be validated by different animal models of type-2 diabetes and human clinical trials in the future.


Introduction
Metabolic syndromes, including obesity, coronary heart diseases, stroke, hyperuricemia, chronic kidney diseases, and diabetes, tend to occur at younger ages and recently have become one of the biggest global health problems, especially in developed and developing countries [1]. Metabolic disorder-related diseases can directly or indirectly interact with other conditions (i.e., obesity dramatically increases the risk of type-2 diabetes [2], or diabetes associated with cardiovascular diseases) [3], thus making their control more difficult. Diabetes is a severe metabolic syndrome closely related to various complications such as chronic kidney disease, blindness [4], and Alzheimer's disease [5]. Numerous drugs are available to control glycemic levels in diabetes patients; however, they usually have various side effects, such as gastrointestinal distress, nausea, and diarrhea, that lower quality of life.
Probiotics are symbiotic microorganisms that reside naturally in the human skin, gut, respiratory tract, genital tract, and mucosal tissues [6]. Their populations are markedly affected by the daily lifestyle of the host, and any consequent changes can cause sub-health conditions such as atopic dermatitis, obesity, asthma, allergic diseases, inflammatory bowel disease, and oral ulcers. The regular human dosage for daily probiotic consumption is around 1 � 10 10 [7].
Chronic low-grade inflammation mediated by M1-type adipose tissue macrophages (CD8 T and Th1 cells) in long-term overweight or obese individuals can result in insulin resistance and type-2 diabetes [8]. Diabetes patients are estimated to account for approximately 8% of the global population [9], of which 95% has type-2 diabetes. Previous studies have shown that the gut microbiota is involved in energy metabolism and associated with metabolic disorders such as obesity and type-2 diabetes; however, this interrelationship is affected by genetic and environmental factors.
We have previously shown that certain probiotic strains reduce blood glucose levels in mice [10]. Therefore, in this study, we selected probiotic strains Lactobaccilus salivarius subsp. salicinius AP-32, L. johnsonii MH-68, L. reuteri GL-104, and Bifidobacterium animalis subsp. lactis CP-9 as major components of a probiotic product and investigated its effects on a type-2 diabetic rat model obtained with STZ treatment and a high-fat diet [11,12]. Glycemic index, insulin levels, beta-cell mass, blood lipid levels, and anti-oxidant activity were determined.

Animals
All experiments and protocols complied with the Laboratory Animal Care and Use Guidelines published by the Taiwan government. The protocols were approved by the Shih Chien University Animal Ethics Committee (Permit no. 10803). Male Sprague Dawley (SD) rats were purchased from BioLasco (Taiwan) and housed at the Laboratory Animal Center, Shih Chien University, Taipei, Taiwan, under controlled conditions (12-h light/12-h dark cycle, 22˚C ± 2˚C, and 62% ± 5% humidity). The animals were provided with sterilized water and food throughout the experimental period (weeks [1][2][3][4][5][6][7][8]. In this experiment, 50 six-week-old male SD rats were purchased from Lesco, originally 10 in each group. At 0 weeks, 2 rats in each group with poor mobility were exclude (e.g. rats with no responding of grasp), and finally 8 male SD rats were in each group. The standard for animal humane suspension of the experiment is to observe the physiological state of the animal every day during the experiment to see if there is any abnormal behavior of weakness, motionlessness, stopping water or eating. If there is any abnormal behavior, the experiment shall be terminated humanely in advance. The groups are: normal control group (Normal Control, N) and diabetic hyperglycemia group as negative control group (Diabetes Mellitus, DM), probiotics 1X group (DM1X), probiotics 5X group (DM5X), probiotics Bacteria 10X group (DM10X). Groups with induced diabetic hyperglycemia include DM, DM1X, DM5X, DM10X.

Streptozotocin-induced diabetes animal model
SD rats were administered with nicotinamide (NA, 30-60 mg kg-1), 15-30 min before treatment with 10-20 mg kg-1 streptozotocin (STZ) to deactivate β-cells in the pancreas. NA and STZ were injected intraperitoneally every 2 d for 8 weeks. To further elicit diabetes, STZtreated rats were fed with a high-fat diet (Research Diets, D12492) [13]. General anesthesia is required for most methods of blood collection in rats, to prevent restraint. Rats were anesthetized with an intraperitoneal injection of Zoletil/Xylazine (20~40mg/kg Z+5-10mg/kg X). Fasting blood glucose and fasting blood insulin levels were monitored every 2 weeks. The blood samples were collected from the tail of rats.

Evaluation of body weight, food consumption, and water intake
Body weights were measured twice a day at 0, 2, 4, 6, and 8 weeks. For evaluation of the daily food consumption, 35 g of fodder was provided, and the remaining was weighed after 24 h. Data on the daily water intake were collected using a measuring bottle. Body weight, food consumption, and water intake observed in STZ and STZ + Probioglu TM groups were compared with the control group.

Oral glucose tolerance test (OGTT)
For OGTT, all groups were orally administered with 1 ml glucose (1 g kg -1 ) at weeks 4 and 8, and their blood glucose and insulin levels were analyzed at 30 min, 60 min, 90 min, 120 min, and 180 min after administration. Blood glucose was detected using a rat glucose assay kit (Randox, UK), whereas insulin concentrations were analyzed using the Rat Insulin ELISA Kit (Mercodia, Sweden). The total glucose area under the curve (AUC) was grouped and calculated at the time periods of 0-30 min, 30-60 min, 60-90 min, 90-120 min, and 120-180 min as described previously [17].

Determination of β-cell mass
The β-cell mass in the pancreas was detected as described previously [18]. Briefly, immunohistochemical staining of pancreatic sections was performed to determine the area of pancreatic islets. Each pancreas sample was sliced into 50 sections (12 μm thick for each section). The areas of pancreatic islets and total pancreas were analyzed by microscopy and quantified by ImageJ (https://imagej.net/ImageJ). To further calculate the total number of pancreatic cells and that of β-cells, hematoxylin and eosin stains were applied. The cell numbers were counted under 200× magnification of an optical microscope. The β-cell mass was calculated as follows: β-cell mass = weight of the pancreas × (number of β-cells/total number of pancreatic cells)/area of the pancreatic section slide.
The experiments were performed by two independent researchers to prevent bias.

Statistical analysis
Statistical analysis was performed using Microsoft Excel and Prism 8 (GraphPad, USA). Data are presented as means ± standard deviation (SD) obtained from two or three independent experiments and collected from eight animals. Differences were identified using one-way analysis of variance in conjunction with the Duncan's new multiple range test (MRT) and considered significant at p < 0.05.

Probioglu TM induced dose-dependent restoration of food consumption and water intake in STZ-treated rats
In this study, type-2 diabetic rats were induced by low-dose STZ (10-20 mg kg -1 ) and a highfat diet. Body weight, food consumption, and water intake were monitored twice daily at weeks 0, 2, 4, 6, and 8 in all groups (Fig 1). The body weight and food consumption of STZ and STZ + Probioglu TM groups were significantly lower than those of the control group  Fig 1A and 1B); however, Probioglu TM treatment led to a dose-dependent restoration of appetite compared to the DM group at week 8 (DM group with STZ treatment only: 20.87 ± 0.5 g; DM10X: 25.79 ± 2.1 g: p < 0.05 � ; Fig 1B). Water intake was significantly higher in the STZ and STZ + Probioglu TM groups compared with the control group at week 8 (Control: 39.91 ± 1.7 mL; DM10X: 98.38± 4.2 mL; p<0.05 � ; Fig 1C). The restoration of water intake was only observed in the STZ + 10× Probioglu TM group ( Fig 1C).

Probioglu TM reduced glycemic levels and partially restored glucose tolerance in STZ-treated rats
Glycemic levels were monitored throughout the experimental period in all groups (Table 1). Compared with the control group, FBG levels increased in all STZ groups at weeks 2-8; at week 8, glycemic were 3-fold higher in the STZ group (DM: 295.6 ± 9.8 mg/dL) compared with the control group (N: 96.7 ± 9.8 mg/dL). However, the rate of increase was significantly slower in the STZ + 10× Probioglu TM group (DM10X at week 8: 220.5 ± 9.3 mg/dL; p < 0.05 � ) than in the STZ group.
OGTT were evaluated at weeks 4 and 8 in all groups (Fig 2). In the control group, blood sugar levels showed small increases at 60 min after the administration of high-dose glucose and decreased at 120 min at weeks 4 and 8. All STZ groups showed relatively high levels of blood sugar before glucose uptake, whereas the level of blood sugar failed to decrease at 60 min after the administration of high-dose glucose at week 4 (Fig 2A). However, OGTT data at week 8 showed that blood glucose levels in the Probioglu TM uptaking group at 180 min returned to the levels at 0 min (DM10X at 0 min: 218.6 ± 5.1 mg/dL; DM10X at 180 min: 215.6 ± 18.5 mg/dL; Fig 2B). Further analysis of the total AUC during OGTT is shown in Fig 2C and 2D.

Probioglu TM reduces blood insulin levels and insulin resistance (HOMA-IR) in STZ-treated rats
Insulin levels were monitored throughout the experimental period in all groups ( Table 2). Compared with the control group, fasting blood insulin levels increased in all STZ groups at weeks 2-8. At week 8, insulin levels were 3-fold higher in the STZ group than in the control group (STZ group: 3.38 ± 0.15 μg L -1 ; control group: 1.08 ± 0.03 μg L -1 ; p < 0.05 � ). However, the rate of increase was significantly lower in the STZ + 10× Probioglu TM group (2.18 ± 0.12 μg L -1 , p < 0.05 � ).

Probioglu TM attenuated β-cell death and increased β-cell mass in STZ-treated rats
Beta-cell death and beta-cell mass was evaluated at the end of the experimental period in all groups (Fig 3). At week 8, pancreatic islets of STZ groups were smaller than those of the control group and also impaired (control: 320.9 ± 10.4 mg; STZ: 35.3 ± 5.4 mg; p < 0.05 � ; Table 2. Insulin levels of rats in each group at weeks 0, 2, 4, 6, and 8.

Probioglu TM improved serum biochemistry indices in STZ-treated rats
Serum biochemistry and bioindicators of oxidative stress were evaluated at the end of the experimental period in all groups (Tables 3 and 4). Compared with the control group, triglyceride (TG) was 6-fold higher in the STZ group but less than 4-fold higher in the STZ + 5× Probioglu TM Table 3). Both HDL and FFA showed no significant differences in any group (Table 3). Urea levels were significantly higher in all STZ groups compared with the control group, and supplementation with Probioglu TM showed only partial improvements in a dose-dependent manner (control: 11.3 ± 5.8 mg dL -1 ; STZ: 44.1 ± 11.6 mg dL -1 ; STZ + 1× Probioglu TM : 38.3 ± 8.2 mg dL -1 ; STZ + 5× Probioglu TM : 26.1 ± 11.7 mg dL -1 ; STZ + 10× Probioglu TM : 21.8 ± 9.3 mg dL -1 ; Table 3).

Discussion
Two main types of diabetes, type 1 and type 2, are caused by relative or absolute insulin insufficiency. Autoimmune attack of insulin-generating pancreatic β-cells leads to the former type, whereas impaired compensation of β-cells leads to the latter type [19]. Two major animal models of type-2 diabetes, the obese and the non-obese, can mimic insulin resistance and β-cell failure [20,21]. Natural mutations, genetic manipulation, and high-fat feeding are used to develop the obese model. Examples of type-2 diabetes animal models owing to defective leptin receptor-induced obesity are Lepob/ob mouse [22], Leprdb/db mouse [23], and Zucker diabetic fatty rat [24], However, more accurate symptoms and complications of human type-2 diabetes are rendered from polygenic models such as KK mice that present severe hyperinsulinemia, insulin resistance, and diabetic nephropathy [25]; the Otsuka Long-Evans Tokushima Fat rat (OLETF) that demonstrates mild obesity, late-onset hyperglycemia, fibrotic islets, and renal complications [26]; and the New Zealand Obese (NZO) mice that exhibit hyperphagia, obesity, leptin resistance, hyperinsulinemia, elevated blood glucose levels, and hyperplastic islets [27]. Type-2 diabetes animal models are also developed when a high-fat diet (58% of the energy derived from fat compared with 11% of the energy derived from fat in a standard diet) is administered for several weeks, thereby leading to significant weight gain associated with insulin resistance and impaired glucose tolerance [28]. In the present study, a high-fat diet accompanied with low-dose STZ was used to develop type-2 diabetic rats with progressive disease symptoms similar to those in humans, including hyperinsulinemic dysglycemia, hepatic fibrosis, pancreatic β-cell dysfunction, late-stage hyperglycemia, dyslipidemia, decreased myocardial glucose utilization, and renal dysfunction [29].
Streptozotocin (STZ) is commonly used for targeting insulin producing beta cells in pancreas to induce hyperglycemia and mimic T2DM disease conditions. Conventionally, a dosage of 50mg/kg is used [30]. Since STZ is toxic to beta cells, this dosage has been shown to result in necrosis and undesirable high beta cell mortality [30]. In order to mitigate toxicity severity and cell mortality (but the beta cells weren't completely damaged), we implemented 10-20mg/kg dosage in every two days for 8 days. This animal model was optimized according to previous study, in which multiple low doses of STZ and high-fat diet (HFD) was followed to induce T2DM [30,31]. Therefore, this optimized method was primarily designed to induce slow and stable damage of beta cells to generate T2DM model rats(S2 Table in S1 Text).
In the present study, we administered different amounts of Probioglu TM to STZ-treated rats to investigate its effects on the regulation of physicochemical parameters related to type-2 diabetes. T2DM induced rats often exhibit the symptoms of polydipsia, polyuria and polyphagia [32]. At present results (Fig 1) clearly shows that the STZ-induced diabetic rats consumed more food, water, which is in line with the previous studies. To explain this phenomenon, hyperglycemia results in escalation of filtered glucose load, causing expulsion of more glucose in urine [33,34]. Hence, hyperglycemia induces production of high glucose containing urine. The loss of body fluid and high blood glucose lead to tissue dehydration. Therefore, the STZ administered rats are likely to generate a higher volume of urine (polyuria) and urinate more often. The deprivation of water in the tissues might make the rats very thirsty (polydipsia) and trigger signals to drink more water for preventing dehydration. It might be the reason of the rise in water consumption over the last two weeks (Fig 1).
Probioglu TM also attenuated STZ-induced β-cell death and increased β-cell mass (Fig 3). It is known that proinflammatory cytokines can cause pancreatic β-cell failure and destruction in diabetic patients [38]. Our probiotic formula may prevent apoptotic destruction of β-cells by diminishing the proinflammatory cytokines TNF-α, IL-1β, and IL-6 ( Table 4).
Previous studies have shown that oxidative stress may lead to the disruption of normal β-cell function: oxidative stress activates the c-J N-terminal kinases pathway to promote β-cell in diabetes [39]. The improved levels of serum anti-oxidative SOD, MDA, and GSH after Probioglu TM administration may lead to β-cell protection.
Ricardo Beltramede Oliveira et al. revealed that High Fat Diet (HFD) intake would impaired tight junction protein structure at early stage of T2DM [40]. Thus, we tested tight junction protein expression in mRNA level by treating Probioglu TM to intestinal Caco-2 cell. The result present that Probioglu TM would significantly elevate mRNA level of Occludin, JAM-A and ZO-2 by comparing to medium control [41]. S1 Fig in S1 Text revealed Probioglu TM would significantly elevated tight junction protein expression in mRNA level (Occludin, JAM-A and ZO-2) in Caco-2 cell model.
Besides, researchers had demonstrated exercise could ameliorate T2DM induced intestinal SFCA concentration decline in mice model [42]. It is reported that probiotic secreting SCFA would improve glycemic control among T2DM patients [43]. Certain probiotic strains including Lactobacillus rhamnosus GG and L. gasseri PA 16/8, Bifidobacterium longum SP 07/3 and B. bifidum MF 20/5 are able to produce acetate and propionate, but can't generate butyrate [44]. In this study, we further tested the SCFA and MCFA levels generated by Probioglu TM . The Probioglu TM consisting of viable probiotic strains AP-32, CP-9, GL-104 and MH-68 were cultured overnight in MRS medium. Collecting supernatants of individual strain then analyzing SCFA and MCFA contents by HPLC [45]. The functional SCFA including acetic acid, propionic acid and butyric acid were detected in individual strain of AP-32, CP-9, GL-104 and MH-68, which may contribute to mediate glycemic index at present study (S1 Table in S1  Text). However, further experiments should validate how probiotic secreting SCFA regulates blood glucose level and protects beta cells in animal model in the future.

Conclusions
Overall, we showed that injections of low-dose STZ 10-20 mg/kg with a high-energy diet could successfully induce hyperglycemia and cause damage to beta cells of the test pancreas. However, the Probioglu TM , containing L. salivarius subsp. salicinius AP-32, L. johnsonii MH-68, and L. reuteri GL-104, alleviated the symptoms of type-2 diabetes in STZ-treated rats by protecting the function of β cells and stabilize the glycemic levels. Additional type-2 diabetes animal models, such as Leprdb/db mouse, NZO mice, and several non-obese animal models, may be needed to validate the benefits of Probioglu TM . In addition, the molecular mechanism of β cell protection and clinical study by Probioglu TM needs further investigation.