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. 2023 Nov 17;33:242–250. doi: 10.1016/j.bioactmat.2023.11.003

Zn-Fe primary battery-enabled controlled hydrogen release in stomach for improving insulin resistance in obesity-associated type 2 diabetes

Boyan Liu a,1, Peixun Lv a,b,1, Xiaoyi Zhang a, Chao Xia c, Xinru Liu a, Jingyu Liu a, Junli Xue a, Qianjun He b,d,∗, Shucun Qin a,∗∗
PMCID: PMC10689207  PMID: 38045569

Abstract

Chronic systemic inflammation in obesity-associated type 2 diabetes (T2D) is a key inducing factor of insulin resistance (IR). Hydrogen molecule (H2) has been proved to be a safe and effective anti-inflammatory agent, but conventional H2 administration methods cannot provide a high dosage and a long duration of H2 treatment in IR-related tissues and thus lead to limited therapeutic efficacies. We here propose a new strategy of controlled H2 release to match the time window of gastric emptying for maximizing the bioavailability and therapeutic outcome of H2. This work enhances the hydrolysis rate of Zn by constructing a Zn-Fe primary-battery micro-/nano-structure, and the H2-releasing rate is adjusted by tuning the ratio of Zn to Fe. The Zn-Fe micro-/nano-structure is orally administrated once daily to alleviate obesity-associated T2D in a leptin-deficient (ob/ob) mouse model. The H2 generation time of the Zn-Fe primary-battery micro-/nano-structure with the Fe/Zn ratio of 1:100 in gastric acid is about 3 h, just matching with the time window of gastric emptying in mice. In vivo monitoring results show that H2 generated by Zn-Fe micro-/nano-structure in stomach can effectively accumulate in major IR-sited tissues including liver, adipose tissue, and skeletal muscle at a high dose for a relatively long time compared to H2-rich water drinking. Oral administration of Zn-Fe micro-/nano-structure at 200 mg/kg body weight has realized an efficient IR improvement and remarkably ameliorated systemic inflammation in ob/ob mice. In addition, a high-dose administration of Zn-Fe shows no visible toxicity in mice. This work provides a new strategy to maximize the outcome of hydrogen therapy.

Keywords: Hydrogen therapy, Primary battery, Type 2 diabetes, Insulin resistance, Anti-inflammation

Graphical abstract

Image 1

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Highlights

  • •

    A new Zn-Fe primary-battery micro/nanostructure is developed to control the H2-generating rate.

  • •

    The H2-releasing time duration matches with the time window of gastric metabolism.

  • •

    H2 generated by Zn-Fe micro/nanoparticles effectively accumulates in insulin resistance-sited tissues.

  • •

    Zn-Fe micro/nanoparticles enhance the anti-IR outcomes of H2 and efficiently alleviate T2D.

1. Introduction

Type 2 diabetes (T2D) is a serious health problem globally and it is estimated that 439 million people would catch T2D by 2030 [1]. It is well known that T2D is a chronic metabolic disorder highly interrelated with obesity. In the progression of obesity-associated T2D, insulin resistance (IR) frequently causes target cells fail to normally respond to insulin [2]. Increasing evidences indicate that chronic systemic inflammation is a key factor of inducing IR. Typically, sustaining inflammation in liver, adipose tissue, and skeletal muscle locally causes IR by inhibiting insulin signal transduction [3]. Thus, it is promising to develop broad-spectrum anti-inflammatory agents targeting multiple IR-sited tissues for effective anti-IR.

Generally, lifestyle interventions for weight reduction are adopt to improve IR, but it is difficult for individuals to insist, and therefore pharmacological management is frequently needed. Current pharmacological agents against IR mainly include metformin (Met) and pioglitazone [4]. However, both drugs have certain side effects in the incident of gastrointestinal adverse events and edema [5,6], so that their anti-IR efficacies are hardly sustainable in a long run [4]. Therefore, there is an urgent need to develop safe and effective anti-IR new technologies.

Hydrogen molecule (H2) has been proved to be a safe, effective and broad-spectrum anti-inflammatory agent which brings benefits for many inflammation-related diseases including metabolic diseases [7,8], cancer [9], and nervous system disorders in clinical researches [10]. Oral administration of H2-rich water (HRW) is found to be able to improve glucose metabolism in T2D patients and animal models [[11], [12], [13]], but the amount of H2 delivered by water is rather low because the saturated solubility of H2 in water is considerably low (0.8 mmol/L at room temperature and normal pressure) and the daily uptake amount of water is limited. The development of solid H2 donors such as metal hydrides, borides and silicides will favor to enhance the efficiency of H2 delivery [[14], [15], [16], [17], [18], [19]]. Several oral agents of H2 donor have been developed to enhance the efficiency of H2 delivery, but there are still many issues. Oral administration of H2 producing materials such as magnesium (Mg) microparticles, magnesium hydride (MgH2) microparticles, calcium hydride (CaH2) microparticles, iron (Fe) nanoparticles and ammonia borane (AB) encapsulated hollow mesoporous silica nanoparticles (AB@hMSN) is convenient and can support a high H2-dose [20], but their H2-releasing rates in gastric acid is either too fast (Mg, MgH2, CaH2, and AB@hMSN) or too low (Fe and Zn) compared with the time window of gastric emptying (about 4 h for human [21] and 3 h for mice [22]). Thus, it is a big challenge to control their H2-releasing rates for matching with the time window of gastric emptying in order to maximize the bioavailability and therapeutic outcome of H2.

Herein, we proposed a primary-battery strategy to construct a new kind of Zn-Fe micro-/nano-structure, realizing the controlled acid-responsive H2 release and adjusting the H2 releasing duration within the time window of gastric metabolism (Scheme 1). In vivo monitoring results showed that H2 generated by Zn-Fe in stomach can effectively accumulate in major IR-sited tissues including liver, adipose tissue, and skeletal muscle at a high dose for a relatively long time. Oral administration of Zn-Fe once daily can ameliorate the obesity-related inflammation, and realize efficient IR improvement in an obese diabetic leptin-deficient (ob/ob) mouse model.

Scheme 1.

Scheme 1

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Schematic illustration for the structure and construction method of the Zn-Fe primary-battery micro-/nano-structure (A), the primary-battery principle for accelerated Zn hydrolysis and H2 generation (B), the efficient H2 accumulation in insulin resistance-sited tissues after oral Zn-Fe administration in support of enhanced anti-inflammation and improved insulin tolerance (C).

2. Material and methods

2.1. Chemicals

Zn powder, ferric chloride (FeCl3) and zinc oxide (ZnO) all were purchased from Sigma-Aldrich. Ethanol was purchased from Energy Chemical.

2.2. Synthesis and characterization of Zn-Fe and ZnO-Fe2O3

The synthesis of Zn-Fe primary battery was based on a displacement reaction. By applying the reducibility of Zn, iron ion was reduced to zero-valent Fe on the surface of Zn microparticles. Details were as follows: 162.2 mg of FeCl3 was dissolved into 50 mL deionized water to form 0.02 mol/L FeCl3 solution. According to the different molar ratios of FeCl3:Zn (10:100, 1:100 and 1:300), 65, 650, and 1950 mg of Zn microparticles were dispersed into 5 mL FeCl3 solution under rapid stirring. After reaction for 10 min, the samples were collected by centrifugation, washed with deionized water and ethanol in turn, and then dried under vacuum to obtain Zn-Fe primary-battery nano/microparticles with different Fe/Zn ratios (Fe:Zn = 10:100, 1:100, and 1:300).

ZnO-Fe2O3 was synthesized as a negative control of Zn-Fe. Fe2O3 was grown on the ZnO surface by hydrothermal synthesis. The details were as follows: 202.75 mg FeCl3 was dissolved in 1 mL deionized water to form 202.75 mg/mL FeCl3 solution, and 5 mg KH2PO4 was dissolved in 1 mL deionized water to form 5 mg/mL KH2PO4 solution. Then, 1.017 g ZnO was dispersed in 30 mL deionized water, and then 100 μL of FeCl3 solution and 100 μL of KH2PO4 solution were added in turn. After ultrasonic dispersion, the above system was transferred to a polytetrafluoroethylene-lining reactor, whose temperature was increased to 150 °C at a heating rate of 3 °C/min and then maintained at 150 °C for 48 h. After cooling, the sample was washed twice with deionized water, collected by centrifugation at 12,000 rpm, and then freeze-dried to obtain ZnO-Fe2O3.

The morphology and size of the Zn-Fe primary battery were characterized by scanning electron microscopy (FEI Apero S). The chemical valence of Zn-Fe primary battery was detected by X-ray photoelectron spectroscopy (XPS). The crystal structure was characterized by X-ray diffraction (PANalytical, Empyrean).

2.3. Measurement of H2 generation in vitro

Different metal powders (Zn, Fe and Mg) and Zn-Fe nano/microparticles with different Fe/Zn ratios (Fe:Zn = 10:100, 1:100, and 1:300) were added into the phosphate buffer solution (PBS) with a pH value of 1.2 to simulate gastric condition at 37 °C. The release of H2 was monitored in real time by gas chromatography (Agilent 7890B) [23].

2.4. Pharmacokinetics study

Six to eight-week-old male wild-type (WT) C57BL/6J mice were intragastrically administered with Zn-Fe (20 mg/mL in water) or HRW (0.8 mmol/L H2 in water) at a dose of 0.1 mL/10 g body weight (BW) (equal to Zn-Fe 200 mg/kg BW). The H2 concentrations in liver, adipose tissue (epididymal fat pad), and skeletal muscle (gastrocnemius muscle) were monitored in real time by a Clark-type H2 sensor (Unisense, Aarhus N, Denmark) [24,25]. At the same time, the serum samples were collected at 0, 1, 2, 3, 6, 12, 24, and 48 h (n = 3). The quantification of Zn concentration in serum was performed using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7800, Agilent Technologies, USA).

2.5. Animal experiments

Six to eight-week-old male WT C57BL/6J mice and C57BL/6J-based ob/ob mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice were housed in a specific pathogen-free facility with a 12/12 h light-dark cycle at 22 °C and given free access to food and water. All animal care and experiments were carried out following protocols approved by the Laboratory Animal Ethics Committee of Shandong First Medical University (No. W202108080304). After 1 week of acclimatization, mice were randomly assigned to six groups (8 mice per group). (Ⅰ) WT group: WT mice intragastrically administered with water at 0.1 mL/10 g BW once daily; (Ⅱ) Model group: ob/ob mice intragastrically administered with water; (Ⅲ) ZnO-Fe2O3 group: ob/ob mice intragastrically administered with aqueous solution of ZnO-Fe2O3 at 200 mg/kg BW once daily; (Ⅳ) Zn-Fe group: ob/ob mice intragastrically administered with aqueous solution of Zn-Fe at 200 mg/kg BW once daily; (Ⅴ) HRW group: ob/ob mice administered with HRW ad libitum (about 1.5 mL HRW, 0.6–1.5 μmol/10 g daily H2 administrated amount) and intragastrically administered with water; (Ⅵ) Met group: ob/ob mice intragastrically administered with aqueous solution of Met at 200 mg/kg BW once daily. The mice received oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) after 11 weeks and were sacrificed after 12 weeks (Fig. S1).

Blood samples were collected in EDTA-coated tubes, centrifuged at 3000 rpm for 15 min at 4 °C, and the plasma was stored at −80 °C. A portion of liver, gastrocnemius muscle, inguinal white adipose tissue, and pancreas were rapidly excised, dipped in liquid nitrogen, and stored at −80 °C for further analysis or fixed in 4% paraformaldehyde for 24 h for histological studies.

2.6. HRW drinking

Mice in HRW group were provided with HRW ad libitum. HRW was prepared in an HRW-producing apparatus (ZQLH-F-150G, Zhongqinglianhe Tech Co., Ltd, Liaocheng, China), and stored in a drinking bottle with stainless steel balls in the outlet. The concentration of H2 in HRW was monitored using a Clark-type H2 microsensor (Unisense, Aarhus N, Denmark). HRW was replaced with freshly prepared one every 8 h to ensure that the concentration of H2 was above 600 μmol/L [26].

2.7. Toxicity assessment

Six to eight-week-old male C57BL/6J mice were randomly divided into four groups (12 mice per group) and intragastrically administered with water (vehicle) or Zn-Fe at 200, 400, and 800 mg/kg BW every day. Six mice in each group were sampled after 1 or 2 weeks. Blood samples were collected for biochemical tests. Major organs including stomach, small intestine, liver, kidney, spleen, and heart were dissected, fixed in 4% paraformaldehyde, and embedded into paraffin for further hematoxylin & eosin (H&E) staining.

2.8. Biochemical analysis

Total cholesterol (TC) and triglyceride (TG) in plasma and liver were measured with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). Plasma levels of insulin, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and c-reactive protein (CRP) were measured using corresponding ELISA kits (Mlbio, Shanghai, China). A bicinchoninic acid protein assay kit (Solarbio Science & Technology Co., Ltd., Beijing, China) was used to determine the protein content in various tissues.

2.9. Oral glucose tolerance test and insulin tolerance test

The OGTT was performed by detecting the blood glucose levels at 0, 15, 30, 60, and 120 min after oral gavage of 2 g/kg glucose to the 12 h fasted mice. The ITT was performed by detecting the blood glucose levels at 0, 15, 30, 60, and 120 min after intraperitoneal injection of 0.75 U/kg insulin to the 4 h fasted mice. The glucose levels were determined in blood drops obtained by clipping the tail of the mice using OneTouch Ultra Mini glucometer (LifeScan; Milpitas, CA, USA).

2.10. Immunohistochemistry and immunofluorescence

Adipose tissue and liver samples embedded in paraffin were stained for expression of F4/80 with an anti-F4/80 monoclonal antibody (ab111101, Abcam, Cambridge, MA, USA, 1:100) according to the standard histological procedure (n = 6 in each group). The immunoreactive cells were counted in five visual field under a 400 × light microscope. Immunofluorescence was performed with primary antibodies, including insulin (gb13121, Servicebio, 1:300) and glucagon (gb11097, Servicebio, 1:200). The percentage of β-cell area in the pancreas was calculated by dividing the area of all insulin-positive cells by the whole area of this section (n = 4 in each group).

2.11. Quantitative real-time PCR

Adipose tissue, skeletal muscle, liver, and pancreas samples were homogenized and total RNA was extracted with TRIzol. The cDNA was produced in an ABI Veriti 96 Well Thermal Cycler (Waltham, MA, USA) using HiFiScript cDNA Synthesis kit from CWBIO (Beijing, China). Quantitative real-time PCR was performed in an ABI QuantStudio3 PCR System (Waltham, MA, USA) using SYBR Green qPCR Master Mix and gene-specific primers. The 2−ΔΔCT method was used to normalize mRNA expression level to that of the endogenous housekeeping gene peptidylprolyl isomerase A (PPIA). This experiment was repeated four times independently. The primer sequences were as follows: TNF-α, 5′-GAACTGGCAGAAGAGGCACT-3’ (forward) and 5′-GGTCTGGGCCATAGAACTGA-3’ (reverse); IL-6, 5′-CCGGAGAGGAGACTTCACAG-3’ (forward) and 5′-TTCTGCAAGTGCATCATCGT-3’ (reverse); monocyte chemoattractant protein-1 (MCP-1), 5′-CCCAATGAGTAGGCTGGAGA-3’ (forward) and 5′-TCTGGACCCATTCCTTCTTG-3’ (reverse); PPIA, 5′-GACCAAACACAAACGGTTCC-3’ (forward) and 5′-CATGCCTTCTTTCACCTTCC-3’ (reverse); insulin receptor substrate 2 (Irs2), 5′-ACAACAACCACAGCGTGCGC-3’ (forward) and 5′-GTAGAGGGCGATCAGGTACTTG-3’ (reverse); insulin receptor (Insr), 5′-AGGCTCCCGTCTCTTCTTCAA-3’ (forward) and 5′-GACATCCCCACATTCCTCGTT-3’ (reverse); pancreatic and duodenal homeobox 1 (Pdx1), 5′-TTCCCGAATGGAACCGAGC-3’ (forward) and 5′-GTAGGCAGTACGGGTCCTCT-3’ (reverse); and insulin like growth factor 1 receptor (Igf1r), 5′-TCCCTCAGGCTTCATCCGCAA-3’ (forward) and 5′-CTTCAGCTTTGCAGGTGCACG-3’ (reverse).

2.12. Transcriptomic analysis

Total RNA was extracted from stomach and small intestine using a Trizol reagent according to the manufacturer's instructions. RNA samples were detected based on the A260/A280 absorbance ratio with a Nanodrop ND-2000 system (Thermo Scientific, USA), and the RNA integrity number was determined by an Agilent Bioanalyzer 4150 system (Agilent Technologies, CA, USA). Paired-end libraries were prepared using a ABclonal mRNA-seq Lib Prep Kit (ABclonal, China) following the manufacturer's instructions. The mRNA was purified from 1 μg total RNA using oligo (dT) magnetic beads followed by fragmentation carried out using divalent cations at elevated temperatures in ABclonal First Strand Synthesis Reaction Buffer. Adaptor-ligated cDNA was used for PCR amplification. PCR products were purified (AMPure XP system) and library quality was assessed on an Agilent Bioanalyzer 4150 system. Finally, the qualified library was sequenced using an Illumina Novaseq 6000 to obtain raw data. Then clean reads were separately aligned to the mouse reference using HISAT2 software (http://daehwankimlab.github.io/hisat2/) to obtain mapped reads. Differential expression analysis was performed using the DESeq2 (http://bioconductor.org/packages/release/bioc/html/DESeq2.html), and differentially expressed genes (DEGs) were identified as |log2(fold change)| > 1 and P-adj <0.05. Gene Ontology (GO) functional classifications and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed using DAVID (http://david.abcc.ncifcrf.gov/home.jsp).

2.13. Statistical analyses

Data were expressed as mean ± SEM unless otherwise indicated. Student's t-test was used to compare the difference between two groups, and one-way ANOVA with Bonferroni's post hoc analysis was used for comparison of more than two groups. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant difference.

3. Results and discussion

3.1. Synthesis and characterization of Zn-Fe micro/nanoparticles

The traditional H2-administration routes of H2-rich water drinking and H2-rich saline injection can only support a relatively low dose of H2, and H2-gas inhalation leads to both a low efficiency of H2 utilization and a high inconveniency for use [26,27]. In recent years, local generation of H2 based on nanomedicines is on the rise [16,28,29]. However, for the disease of multi-organ targets such as IR, there are currently no nanomedicine strategy for sufficient and sustained H2 release in multiple target tissues. Meanwhile, acid-responsive H2 donors such as Mg, MgH2, CaH2, Fe, and Al have been developed, but there are still various deficiencies. In the gastric acid environment, the acid hydrolysis of Mg, MgH2 and CaH2 is too rapid to ensure the in-time and full gastric absorption of generated hydrogen gas; both Zn and Fe are too inactive to ensure their efficient utilization for hydrogen generation in stomach; and a high concentration of Al has latent toxic effects on the human body. Therefore, this work proposed to enhance the acid-hydrolysis rate of Zn by constructing a Zn-Fe primary-battery structure, and to adjust the acid-hydrolysis rate of the Zn-Fe primary-battery by tuning the ratio of Zn to Fe for matching with the time window of gastric metabolism.

The Zn-Fe primary battery was prepared using Zn microparticles and FeCl3 as reactants by a displacement reaction. SEM and corresponding element mapping images (Fig. 1A and B) showed that iron nanoparticles were uniformly coated on the surface of zinc particles. Owing to local displacement/deposition, the Fe coating was so stable that it cannot be removed by washing, which is beneficial to form a steady primary-battery structure. Furthermore, XPS patterns of Zn-Fe primary battery suggested that iron was reduced to a zero-valence state on the surface of Zn, confirming the primary battery structure definitely (Fig. 1C and D). By the same displacement method, three samples with different ratios of Fe to Zn were prepared to investigate the effect of Fe/Zn ratio on the rate of H2 generation. From Fig. 1E, the primary-battery structure can indeed accelerate the hydrolysis of Zn for H2 generation in a simulated gastric acid solution, and the higher ratio of Fe to Zn caused faster H2 generation because more primary-battery microstructures were formed between more anodes (Fe nanoparticles) and a cathode (Zn microparticle) (Scheme 1B). From SEM images in Fig. S2, high amounts of Fe/Zn displacement led to the surface corrosion of Zn microparticles to form a porous structure, which might favor the acid hydrolysis of Zn-Fe primary-battery micro/nanoparticles.

Fig. 1.

Fig. 1

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The representative SEM image (A) and corresponding EDS element mapping (B) of Zn-Fe micro/nanoparticles, XPS spectra of Zn-Fe micro/nanoparticles (C, D), and acid-responsive H2 generation profile of Mg, Fe, Zn, and Zn-Fe micro/nanoparticles with different Fe/Zn ratios in the simulated gastric acid solution (E). The scale bar in figure A corresponds to 5 μm.

By comparison, Mg microparticles almost completely decomposed within 5 min in the simulated gastric acid solution (Fig. 1E). Release time duration was so short that the generated H2 gas can be quickly eructated instead of fully absorbed by stomach. On the other hand, the H2 generation of both Zn and Fe microparticles was too slow (Fig. 1E). Only when the ratio of Fe to Zn in the Zn-Fe primary battery was 1:100, the time of H2 generation in the simulated gastric acid solution was just 3 h (Fig. 1E), which can match with the average time period of gastric emptying. Therefore, the Zn-Fe primary battery with this Fe/Zn ratio was used for following therapeutic experiments. Even though there is a different in the time window of gastric metabolism between mice and human being, we can still ensure the match of the acid-hydrolysis rate of the Zn-Fe primary-battery micro-/nano-structure with the time window of gastric metabolism in human being by tuning the ratio of Fe to Zn. Theoretically, the H2-producing rate of the Zn-Fe primary battery can achieve 30.8 mg/g (375 mL/g) after complete acidolysis, which is far higher than the H2-loading capacity of saturated HRW (1.6 μg/g). As a control without H2 generation, the mixture of ZnO and Fe2O3 with the same Fe/Zn ratio (1:100) was prepared by a co-precipitation method. The compositions of Zn-Fe and ZnO-Fe2O3 were further confirmed by XRD characterization (Fig. S3 and Fig. S4). Additionally, we detected the Zn level in the blood of mice after intragastric administration of Zn-Fe micro/nanoparticles with the Fe/Zn ratio of 1:100 (200 mg/kg) to clarify the in vivo metabolic profile. As shown in Fig. S5, the level of Zn in the blood rapidly increased within the first 3 h, and then decreased rapidly to the normal level after 6 h, which was in good accordance with the Zn-Fe hydrolysis progress in stomach and also implied quick metabolism of decomposition products in the body.

3.2. H2 concentration monitoring in vivo

As a gas molecule with low solubility in water, H2 gas is characterized with relatively slow dissolution (about 30 min) and exsolution (several hours) rates. Therefore, the sustained H2 release behavior of the developed Zn-Fe primary-battery micro/nanoparticles were expected in favor of achieving a high bioavailability of H2 in the stomach. The in vivo dynamic curves for H2 concentrations in the stomach wall and three IR-related tissues of interest (liver, adipose tissue, and skeletal muscle) were locally detected in real time by a hydrogen microelectrode after intragastric administration of Zn-Fe micro/nanoparticles with the optimal Fe/Zn ratio of 1:100 (200 mg/kg). Meanwhile, oral administration of freshly-prepared saturated HRW (0.8 mM, 10 mL/kg BW) was used as a control to check the advantages of the Zn-Fe primary-battery micro/nanoparticles.

H2 concentration in the stomach wall quickly exceeded 200 μmol/L in 10 min (Fig. S6), possibly because H2 generation from the acid hydrolysis of Zn-Fe occurred in the stomach. In general, H2 concentrations rose rapidly, reached the highest values of 95.8, 102.0, and 1.7 μmol/L in liver (Fig. 2A), adipose tissue (epididymal fat pad) (Fig. 2B), and skeletal muscle (gastrocnemius muscle) (Fig. 2C) in 20.0, 15.0, and 7.5 min after Zn-Fe administration, respectively. Then H2 concentrations decreased gradually, and still retained over 13.8%, 15.1% and 6.1% of the highest value after 6 h. Such high H2 concentration and retention duration in investigated tissues, especially liver and adipose tissue, should be attributed to a high H2-generating capability of Zn-Fe and a higher solubility of H2 in fat than in water (high lipophilicity). Inhalation of H2 gas has been widely investigated in basic and clinical studies, but it is not convenient for practical use and H2 concentration in the liver quickly decreased by 90% in 5 min after H2 gas inhalation was suspended [24,25]. In comparison, oral administration of Zn-Fe seems more convenient and effective.

Fig. 2.

Fig. 2

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In vivo monitoring of H2 concentration in liver (A, D), epididymal fat pad (B, E), and gastrocnemius muscle (C, F) after intragastric administration with Zn-Fe micro/nanoparticles with the Fe/Zn ratio of 1:100 (200 mg/kg) (A‒C) or HRW (800 μM, 10 mL/kg) (D‒F).

Compared with Zn-Fe micro/nanoparticles, intragastric administration of HRW supplied a remarkably lower H2 concentration and a shorter duration of H2 in various tissues. In the liver (Fig. 2D), adipose tissue (epididymal fat pad) (Fig. 2E), and skeletal muscle (gastrocnemius muscle) (Fig. 2F), the highest H2 concentrations were only 3.0, 4.3, and 0.45 μmol/L in 6.3, 4.5, and 4.5 min after HRW administration, and H2 concentrations decreased by about 95% in 1 h in these three tissues. The speed to achieve the peak value of H2 concentration in tissues was faster compared with Zn-Fe administration, possibly because HRW saved the dissolution time of H2 gas. But the peak value of H2 concentration in tissues was much lower and the half-life time of H2 in tissues was also remarkably shorter compared with Zn-Fe administration, possibly owing to the lower H2 dose supplied by HRW. Taking together, the above results indicated that oral administration of the developed Zn-Fe primary-battery micro/nanoparticles can ensure a high H2 concentration and a long retention duration in major IR-sited tissues and therefore might be especially suitable to anti-IR in obesity-associated T2D.

3.3. Sustained H2 supply ameliorates obesity and hyperlipidemia in ob/ob mice

To examine the therapeutic potential of Zn-Fe primary-battery micro/nanoparticles in obesity-associated T2D, we used leptin-deficient ob/ob mice as a model to mimic the conditions of obesity and T2D development [30], while wild-type C57 mice (WT) without development of obesity and T2D were used as a normal control. As demonstrated in Fig. S1, oral administration of Zn-Fe (200 mg/kg), ZnO-Fe2O3 (200 mg/kg), HRW (0.8 mM, 10 mL/kg), and Met (200 mg/kg) once daily were set as therapeutic group, non-hydrogen control group, HRW control group, and Met control group, respectively, to compare the therapeutic outcome of Zn-Fe with traditional therapies. The body weight of mice was monitored every week, and several key parameters including total cholesterol (TC) and triglyceride (TG) levels in plasma and liver, the dimension of adipose cells and the liver/body weight ratio were measured at the end of treatment (12 weeks) to evaluate therapeutic outcomes.

From Fig. 3A, it can be found that the mice body weight of model group increased much faster than that of WT group, reflecting the successful model establishment. At the end of treatment, compared with the model group, Zn-Fe and Met treatments reduced body weight by 6.9% and 4.1%, respectively, whereas HRW treatment had no significant effect on body weight (Fig. 3A). Meanwhile, there was no significant difference in the food uptake amount of ob/ob mice between treatment groups and the control group (Fig. S7). Both Zn-Fe and HRW treatments significantly decreased plasma TC levels (Fig. 3B), but only Zn-Fe treatment significantly decreased plasma TG (Fig. 3C). Compared with model group, there was a significant reduction of adipocyte size in inguinal white adipose tissue in Zn-Fe, HRW and Met groups, which was quantitatively reflected as the dimension of adipose cells per field (Fig. 3D and E). Both Zn-Fe and HRW effectively reduced the liver/body weight ratio of mice (Fig. 3F and G). In addition, both the number of round fat vacuoles and the swelling of hepatocytes in the liver tissue of mice were clearly reduced by Zn-Fe treatment (Fig. 3H). Supporting this, the hepatic TC (Fig. S8) and TG (Fig. S9) values were also lowered by both Zn-Fe and HRW treatments. ZnO-Fe2O3, which cannot generate H2 and served as a negative control, showed no functional effect. Metformin (Met), which was used as positive control, resulted in a similar therapeutic effect with Zn-Fe treatment. But Zn-Fe can bring more benefit for body weight reduction than Met. By comparison with HRW, Zn-Fe can more significantly reduce both the dimension of adipose cells and the body weight of ob/ob mice. Taken together, these results indicated that Zn-Fe can effectively ameliorate obesity, reduce plasma/hepatic lipid levels, and decrease lipid accumulation in both adipose tissue and liver, possibly owing to sustained H2 release, high-concentration H2 accumulation and long-term H2 retention in these target tissues.

Fig. 3.

Fig. 3

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Sustained H2 supply ameliorates obesity and hyperlipidemia in ob/ob mice. Body weight change during treatment (A), plasma TC (B, n = 8) and TG (C, n = 8) levels, representative photographs of HE-stained epididymal fat tissue (D), the average number of adipocytes (E, n = 3), representative photographs of liver (F), and corresponding liver/body weight ratio (G, n = 8). The scale bars in figure D, F and H correspond to 100 μm, 1 cm and 100 μm, respectively. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

3.4. H2 improves glucose tolerance in ob/ob mice

The ob/ob mice will develop severe insulin resistance (IR) with hyperglycemia and hyperinsulinemia, and therefore the therapeutic efficacy of Zn-Fe on glucose homeostasis was evaluated in the present study. For the fasting blood glucose level, Zn-Fe treatment performed best and decreased it by 25.8% compared with the model group (Fig. 4A). Meanwhile, both Zn-Fe and HRW led to a marked improvement in glucose tolerance (Fig. 4A‒C). IR is a situation in which insulin sensitivity is reduced. Although neither Zn-Fe nor HRW revealed a significant effect on insulin tolerance (Fig. 4E and F), both of them significantly decreased the plasma insulin level of ob/ob mice compared with the model group (Fig. 4D). Especially, the insulin-inhibiting effect of Zn-Fe was remarkably bigger than HRW, resulting in a 42.7% reduction in insulin level (Fig. 4D). Compared with WT, the ob/ob mice in the model group had large pancreatic islets and the population of insulin producing β-cells (red fluorescence) distinctly increased (Fig. 4G, Fig. S10). After treatment with Zn-Fe or HRW, islet dimension obviously became smaller and the population of β-cells also clearly decreased (Fig. 4G, Fig. S10). Consistently, Zn-Fe treatment remarkably decreased the mRNA levels of β-cell hyperplasia activators including Irs2, Insr, Pdx1, and Igf1r (Fig. S11). This indicated that H2 can play a compensatory function of insulin islet cells when their function gradually lost in diabetes. Collectively, these data suggested that Zn-Fe treatment could improve glucose tolerance in ob/ob mice.

Fig. 4.

Fig. 4

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Sustained H2 supply improves blood glucose homeostasis. Blood fasting glucose levels (A, n = 6), oral glucose tolerance test (OGTT) results (B), area under curve in OGTT (C, n = 6), plasma insulin levels (D, n = 6), insulin tolerance test (ITT) results (E), area under curve in ITT (F, n = 6), immunohistochemical staining of pancreatic sections to detect the expressions of insulin (red) and glucagon (green) (G). The scale bar in figure G corresponds to 100 μm. Data are shown as mean ± SEM, n = 6 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

3.5. H2 ameliorates systemic and tissue proinflammatory responses

Growing evidences have revealed that low-grade systemic inflammation is one of the early and main pathological events that lead to the evolution of IR. H2 has been proven to exert anti-inflammatory effects in multiple basic and clinical studies. In this study, we explored the effect of Zn-Fe treatment on systemic inflammation in ob/ob mice. As expected, the plasma levels of key inflammatory cytokines, IL-6 (Fig. 5A), IL-1β (Fig. 5B), TNF-α (Fig. 5C), and CRP (Fig. S12), were markedly lower in the Zn-Fe group than that in the model group, indicating that systemic inflammation was attenuated by Zn-Fe. Notably, Zn-Fe appeared to work better than HRW. IR and obesity are related to chronic inflammation in metabolic tissues. In this research, Zn-Fe significantly decreased the mRNA expressions of MCP-1 and TNF-α in liver (Fig. 5D), adipose tissue (Fig. 5E), and skeletal muscle (Fig. 5F). Considering the critical role of the activated tissue resident macrophages as a source of cytokines in IR, we next performed immunohistochemical analysis with antibody against F4/80, a macrophage marker. Results showed that macrophage infiltration in liver (Fig. 5G) and adipose tissue (Fig. 5H) was significantly reduced by Zn-Fe treatment compared to the model group. Taken together, these results confirmed that H2 generated by Zn-Fe in stomach suppressed systemic and tissue inflammation in ob/ob mice.

Fig. 5.

Fig. 5

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Sustained H2 supply alleviates systemic and local inflammatory response. Plasma levels of IL-6 (A, n = 8), IL-1β (B, n = 8), and TNF-α (C, n = 8) after various treatments, relative mRNA expressions of MCP-1 and TNF-α in liver (D, n = 4), adipose tissue (E, n = 4), and skeletal muscle (F, n = 4), immunohistochemical analysis of F4/80 in liver (G) and adipose tissue (H). The scale bars in figure G and H correspond to 100 μm. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

3.6. Transcriptomic analysis of stomach and small intestine

As Zn-Fe reacted with gastric acid in the stomach and then generated H2 will be absorbed by gastrointestinal tract, we performed transcriptomic analysis of stomach and small intestine to reveal the potential impact of oral Zn-Fe administration. For the stomach, a total of 1065 genes were significantly upregulated and 837 genes were significantly downregulated in the Zn-Fe group compared to the model group (Fig. S13). Gene Ontology (GO) analysis indicated that differentially expressed genes (DEGs) were mainly associated with keratinization, ion transport, fatty acid transport, and lipid metabolic process, etc. (Fig. S14). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the enriched pathways of DEGs in the stomach were mainly related to gastric acid secretion, fat digestion and absorption, cAMP signaling pathway, and vascular smooth muscle contraction, metabolic pathways, insulin secretion, etc. (Fig. S15). Meanwhile, Zn-Fe did not show an obvious metabolism-related effect on the small intestine compared to the model group (Fig. S16‒S18), possibly because H2 production by the acid-hydrolysis of Zn-Fe occurred in the stomach rather than in the small intestine. Taking together, these results suggested that besides a high dose of H2 towards IR-sited tissues for inflammation scavenging and anti-IR, oral administration of Zn-Fe can also directly affect the lipid metabolic function of stomach in ob/ob mice.

3.7. In vivo safety evaluation

As a new oral formula, the biosafety of Zn-Fe micro/nanoparticles deserves attention. To evaluate the safety of Zn-Fe micro/nanoparticles, C57BL/6J mice were intragastrically administered with blank water or different doses of Zn-Fe micro/nanoparticles with the Fe/Zn ratio of 1:100 (200, 400, and 800 mg/kg) every day. First, there were no significant changes in body weight (Fig. S19) and food-intaking amount (Fig. S20) of the mice treated with various doses of Zn-Fe for 1 or 2 weeks compared with the blank control. Moreover, key plasma indicators for liver (ALT, AST, and ALP) (Fig. S21) and kidney (Cr and BUN) (Fig. S22) functions were examined 1 and 2 weeks after oral Zn-Fe administration. All these biochemical assay data were normal at all investigated dosages of Zn-Fe. In addition, there were no histopathological abnormalities in the stomach, small intestine, liver, kidney, spleen and heart of Zn-Fe treated mice (Fig. S23 and Fig. S24). Noticeably, Fe and Zn are two essential trace element and play a key role for some important biological processes. Considering that a high dose administration of Zn-Fe showed no visible toxicity, it is possible for oral Zn-Fe administration to become an efficient and convenient strategy in clinical use.

4. Conclusions

In summary, we developed a kind of Zn-Fe primary battery micro/nanoparticles with an accelerated acid-responsive H2 release behavior in the stomach by the primary-battery reaction. By adjusting the proportion of Zn and Fe, we controlled the H2 release rate and realized the sustained H2-release within the time window of gastric metabolism. Oral administration of Zn-Fe micro/nanoparticles caused a relatively high H2 concentration and a long retention of H2 in several IR-related tissues including liver, skeletal muscles and white adipose tissues. We found that the inflammation of these insulin sensitive tissues was suppressed, and IR was improved in the obesity-associated type 2 diabetic ob/ob mice. Meanwhile, oral administration of Zn-Fe micro/nanoparticles showed a high safety profile. These advantages make Zn-Fe micro/nanoparticles hold a high potential clinical translation.

Data availability

Data will be made available on request.

CRediT authorship contribution statement

Boyan Liu: Investigation, Methodology, Writing – original draft. Peixun Lv: Investigation, Methodology, Writing – original draft. Xiaoyi Zhang: Investigation. Chao Xia: Investigation, Methodology. Xinru Liu: Investigation. Jingyu Liu: Investigation. Junli Xue: Investigation. Qianjun He: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing. Shucun Qin: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare no conflicts of interest regarding the publication of this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China [82172078, 81770855, 82200508]; Academic Promotion Programme of Shandong First Medical University [2019QL010]; National Key Research and Development Program of China [2022YFB3804500]; Shenzhen Science and Technology Program [RCJC20210706092010008]. We thank Figdraw (www.figdraw.com) for providing scientific drawing materials.

Footnotes

Peer review under responsibility of KeAi Communications Co., Ltd.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioactmat.2023.11.003.

Contributor Information

Qianjun He, Email: nanoflower@126.com.

Shucun Qin, Email: scqin@sdfmu.edu.cn.

Appendix A. Supplementary data

The following is the supplementary data to this article:

Multimedia component 1
mmc1.pdf (3.2MB, pdf)

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Associated Data

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Supplementary Materials

Multimedia component 1
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Data Availability Statement

Data will be made available on request.

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