The new dual hormone drug DA5-CH works better than tirzepatide and exendin-4 in a Parkinson’s rat model

Exendin-4 and Tirzepatide had been purchased from Macklin Reagent (China), and DA5-CH was synthesized by GL Biochem (China). Peptides were reconstituted in ultrapure water to a concentration of 1 mg/ml, and aliquots were prepared and stored at -20°C. The following antibodies were used in western blot assay or Immunohistochemistry (IHC): anti-tyrosine Hydroxylase antibody (1: 500; Boster, China), anti-α-Synuclein antibody (1: 500; Boster, China), anti-GFAP antibody (1: 200; Boster, China), anti-IBA-1 antibody (1: 200; Boster, China), anti-TNF-α antibody (1: 200; Boster, China), Anti-GAPDH (1: 2000; Boster, China), secondary antibodies (1: 5000; Boster, China) and a peroxidase-conjugated goat anti-rabbit secondary antibody (1: 200; Boster, China). The following reagents were used in Quantitative reverse transcription (qRT) PCR: SweScript All-in-One RT SuperMix for the qPCR Kit (Servicebio, China), gDNA Remover Kit (Servicebio, China), and 2×Universal Blue SYBR Green qPCR Master Mix Kit (Servicebio, China). Other reagents include 6-hydroxydopamine hydrobromide (6-OHDA, Sigma, USA), pargyline hydrochloride (MedChemExpress, USA), desipramine hydrochloride (MedChemExpress, USA), apomorphine (Absin, China), paraformaldehyde (PFA, Boster Biotechnology, China), Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, China), phenyl-methylsulfonyl fluoride (PMSF, Beyotime, China), BCA Protein Assay Kit (Boster, China), ECL- enhanced chemoluminescence Kit (Boster, China).

All animal experiments were conducted strictly with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Moreover, the ethics board of the Henan Academy approved all experimental procedures. This study utilized 50 three-month-old adult male Sprague-Dawley (SD) rats weighing 220–250 g and sourced from the Experimental Animal Center of the university. All animals had ad libitum admission to water and a standard rat diet and were housed in the animal room under a precisely regulated 12-hour light/12-hour dark cycle. Environmental conditions were maintained at 23 °C ± 2 °C and a humidity level of 55% ± 10%. Thirty minutes before the stereotactic surgery, the rats were administered pargyline (5 mg/kg, a monoamine oxidase inhibitor) and desipramine (10 mg/kg, a noradrenaline uptake inhibitor). Following the induction of general anesthesia via intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight), the rats were securely positioned in a standard stereotaxic apparatus (RWD, China). A single injection of 6-OHDA (5 μl, 2 mg/ml, containing 0.05% ascorbic acid) was carefully delivered into the right medial forebrain bundle (MFB) at the coordinates: A = −2.2 mm anterior to bregma, L = −1.5 mm lateral to bregma, and V = –8.0 mm ventral to the dura. After the injection, the syringe was left in place for 10 minutes to allow for proper diffusion before being slowly withdrawn. Subsequently, the rats were randomly assigned to one of five groups, with each group consisting of 10 rats. Group sizes were estimated according to previous studies that utilized these techniques. Animals were randomized across cages to avoid group effects. The groups were as follows: the sham + saline group, the 6-OHDA + saline group, the 6-OHDA + Exendin-4 group, the 6-OHDA + Tirzepatide group, and the 6-OHDA + DA5-CH group. For the groups in the 6-OHDA + drug category, the rats were intraperitoneally injected with 10 nmol/kg body weight of the respective drug once daily for 30 days (as seen in Figure 1). In contrast, the sham group received an equivalent volume of saline.

The test was conducted thirty days after the surgery, when the rats had achieved complete postsurgery recovery. Rats were given a subcutaneous injection of apomorphine (0.05 mg/kg), placed individually in a square open Beld (diameter: 75 cm), and tracked using a computer tracking system (EthoVision XT software, Noldus Information Technology, Netherlands) for 30 min. Completed contralateral rotations were recorded by two examiners that were blinded to animal status. Prior to the behavioral test, animals were habituated to the test room overnight.

The open-field test measures spontaneous activity, which is reduced by impaired dopaminergic synaptic transmission. The apparatus was composed of a 75cm diameter square area surrounded by a 35cm high wall. The rat was placed in the center of the open field and its movements were observed for 10 min. The distance of travel and tracks were measured by a computer tracking system (EthoVision XT software, Noldus information technology, Netherlands). After each run, the open field was cleaned with a 70% ethanol solution to prevent the build-up of olfactory cues.

Brain tissues were carefully homogenized and lysed in ice-cold RIPA lysis buffer supplemented with phenyl-methylsulfonyl fluoride (PMSF). The lysates were centrifuged at 12,000 revolutions per minute (rpm) for 20 minutes at 4°C. The protein concentration of the total lysates was accurately determined using the BCA Protein Assay Kit. Equal amounts of protein were loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. After electrophoresis, the proteins were efficiently transferred to polyethylene difluoride (PVDF) membranes. These PVDF membranes were blocked by incubating in a solution containing 5% bovine serum albumin (BSA) for 1 hour. After blocking, the membranes were incubated overnight at 4°C with primary antibodies diluted in the blocking solution (5% BSA). Subsequently, the membranes were incubated with secondary antibodies at room temperature for 1 hour. After this incubation, the membranes were imaged using the ECL-enhanced chemoluminescence Kit. Finally, the western blot images were captured with a chemiluminescent imaging system (Sagecreation, Beijing, China). The intensity of each band was quantified by optical densitometry using Image-Pro Plus 6.0 software (Media Cybernetics, USA), allowing for a detailed and quantitative analysis of the protein expression levels.

Striatal dopamine levels were estimated using a Rat Dopamine ELISA Kit (USCN Life Science Inc., Wuhan, China). All procedures were done according to the manufacturer’s instructions. All samples were diluted (1:2) and added in triplicate to the microtiter plate wells coated with the antibody specific to DA and the plates were incubated for 2h at 37 °C. After washes, a biotin conjugated antibody was added to the wells and the plates incubated for 1h at 37 °C. After washing, wells were incubated with an avidin-conjugated HRP for 30min at 37 °C. Finally, a stop solution was added to stop the reaction, and the color changes were quantified at 450 nm using the BioTek Elisa Reader ELx808. The results were expressed as pg/mg protein.

Total RNA was isolated from brain tissues using TRIzol (Takara, Japan). The concentration and purity of the extracted RNAs were measured with a Spectrophotometer (Thermo Fisher Scientific, USA). Samples with an absorbance ratio (A260/A280) between 1.8 and 2.0 were deemed appropriate for subsequent experiments. cDNA synthesis was carried out using the SweScript All-in-One RT SuperMix for the qPCR Kit and gDNA Remover Kit, following the manufacturer’s precise protocol. For the amplification of the synthesized cDNA, specific primers were employed in a 10 μl PCR reaction system with the 2×Universal Blue SYBR Green qPCR Master Mix Kit. The primer pairs utilized are presented in Table 1. Gene expression analysis was conducted on a QuantStudio™ 6 Flex Realtime-PCR system (Thermo Scientific, USA) using 96-well plates. The reaction conditions included an initial denaturation step at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 15 s, annealing/extension at 60°C for 30 s. The total reaction volume was 15 μl. Data analysis was conducted using the 2-ΔΔCt method to calculate the relative ratio of the target gene to the housekeeping gene.

After thirty days post-surgery, the rats were sacrificed and underwent transcardial perfusion with a 4% paraformaldehyde (PFA) solution. Following overnight post-fixation in the same fixative, the brains were gradient dehydrated with ethanol and xylene for cryoprotection. Subsequently, the brains were embedded with paraffin and sectioned at a thickness of 4 µm using a cryostat (Leica, Germany). The substantia nigra pars compacta (SNpc), and striatum sections were cut according to the rat brain atlas by George Paxinos and Charles Watson (sixth edition). These sections were collected and stored at 4°C. For immunohistochemistry, the sections were placed in a 5% hydrogen peroxide (H₂O₂) solution in PBS for 15 minutes to quench endogenous peroxidase activity. After being washed, the sections were successively treated with a 0.25% Triton-X 100 solution in PBS, followed by blocking with 5% goat serum for 1.5 hours. Then, they were immunostained with specific primary antibodies overnight at 4°C. After two washes in PBS, the sections were incubated with a peroxidase-conjugated goat anti-rabbit secondary antibody for 2 hours in the dark at room temperature. Finally, the sections were observed under a microscope (Olympus BX51, Japan). After digitalizing the images, counting was manually performed using the Image-Pro Plus 6.0 software.

Statistical analysis was performed using GraphPad Prism 10 software, and data were expressed as the mean ± standard deviation (SD). Differences among all groups were determined using a one-way analysis of variance (ANOVA) followed by a Tukey multiple comparison test. P < 0.05 was considered statistically significant.

We tested apomorphine-induced rotational behavior to evaluate the functional effects of the lesions and the drug improvements. Ten minutes after the APO injection, the rats rotated with their healthy hind limb (left hind limb). As shown in Figure 2B. Compared with the sham-surgery group, the number of rotations in the 6-OHDA model group increased significantly, indicating that the model was successfully established. After drug treatment, the number of rotations decreased significantly, indicating that drug treatment could reduce the number of rotations of 6-OHDA-induced PD rats. Among them, the therapeutic effect of DA5-CH was better than that of Exendin-4, and Exendin-4 was better than that of Tirzepatide (see Figure 2A). The one-way ANOVA showed a significant difference (F(4,25)=67.83; p<0.0001) (see Table 2).

General spontaneous activity was measured using a computer tracking system to evaluate if spontaneous activity has gone down after 6-OHDA treatment. The results show that all drugs were able to improve spontaneous activity. DA5-CH was more effective than Exendin-4 and Tirzepatide (see Figure 2B; Table 3).

The one-way ANOVA showed a significant difference (F(4,25)=44.2; p<0.0001).

Incretin analogues protect dopaminergic neurons in the substantia nigra against 6-OHDA.

Structurally similar to dopamine, 6-OHDA is a neurotoxic compound that can selectively impair dopaminergic neurons, resulting in a substantial loss of these neurons in the substantia nigra (SN). Consequently, it can mimic both the pathological and behavioral features of PD. Tyrosine Hydroxylase (TH) is a crucial enzyme in catalyzing the conversion of tyrosine into L-DOPA (L-3,4-dihydroxy-phenylalanine). Since L-DOPA is a precursor for dopamine synthesis, the expression level of TH directly reflects the functional status of dopaminergic neurons. To assess this dopamine-related injury, we employed immunohistochemistry (IHC) to stain TH in the substantia nigra to quantify the TH expression.

As shown in Figure 3A, immunohistochemical analysis of TH-positive dopaminergic neurons in the substantia nigra revealed a drastic reduction in cell number in the 6-OHDA-lesioned group compared to the sham-surgery group, indicating severe dopaminergic neurodegeneration. Treatment with DA5-CH significantly mitigated this neuronal loss. Exendin-4 showed some improvements, while Tirzepatide did not show any significant improvement (see Table 4).

The one-way ANOVA showed a significant difference (F(4,25)=87; p<0.0001).

To evaluate how much dopamine levels are affected by the reduction of TH expression, we measured dopamine levels in the striatum. 6-OHDA treatment reduced levels significantly, and DA5-CH was able to normalize dopamine levels. DA5-CH was superior to Tirzepatide and Exendin-4 (see Figure 3B; Table 5).

The one-way ANOVA showed a significant difference (F(4,25)=35; p<0.0001).

Glial fibrillary acidic protein (GFAP) is crucial in constructing the astrocyte cytoskeleton and is exclusively expressed upon astrocyte activation. To assess the activation status of astrocytes, we employed IHC staining of GFAP.

As shown in Figure 4A, our findings revealed that compared to the sham-surgery group (Sham Saline), the 6-OHDA model group (6-OHDA Saline) exhibited a dramatically elevated number of GFAP(+) astrocytes, indicating robust astrocyte activation induced by 6-OHDA. Following treatment with DA5-CH, there was a significant reduction in GFAP(+) cell count, demonstrating effective inhibition of astrocyte activation. Exendin-4 was more effective than Tirzepatide, but less effective than DA5-CH (see Table 6).

The one-way ANOVA showed a significant difference (F(4,25)=31; p<0.0001).

To assess the neuroinflammation triggered by 6-OHDA and the influence of drug treatment on microglial activation, we chose IBA-1, a protein uniquely expressed in microglia that acts as a specific indicator of microglial activation. Subsequently, we measured the expression level IBA-1 in the striatum through IHC staining. As shown in Figure 4B, our findings indicate that compared to the sham-surgery group, the 6-OHDA model group exhibited a dramatically elevated number of IBA-1(+) microglia, reflecting robust microglial activation induced by 6-OHDA. DA5-CH was able to reverse or prevent this activation completely. Exendin-4 showed a reduced effect, but was more effective than Tirzepatide (see Table 7).

The one-way ANOVA showed a significant difference (F(4,25)=12; p<0.0001).

Interleukin (IL) and TNF-α serve as the central drivers of neuroinflammation. They potentially intensify the inflammatory cascade and cause neuronal damage by activating astrocytes and microglia. To assess the impact of cytokines, we employed RT-qPCR and Western blot assay to quantify the expression levels of IL-6 and TNF-α. During the RT-qPCR analysis, as shown in Figure 5, our findings revealed a significant upregulation of IL-6 and TNF-α in the 6-OHDA +Sal group compared to the sham-surgery group. However, following treatment with drugs, we observed a notable suppression in the elevated levels of these inflammatory cytokines. Specifically, for IL-6, a highly significant reduction was noted, and for TNF-α, a significant decrease was also evident. At the same time, we obtained similar results in the Western blot assay. As shown in Figure 5C, our findings revealed a significant upregulation of TNF-α in the 6-OHDA model group compared to the sham-surgery group (P < 0.001). Treatment with DA5-CH was very effective, followed by Exendin-4 that showed some effects also. Tirzepatide was almost ineffective. It showed no effect in the TNF-α PCR quantification and only a small effect in the IL-6 and TNF-α WB evaluation (see Figure 5; Tables 8–10).

TNF-α PCR results: The one-way ANOVA showed a significant difference (F(4,25)=12; p<0.0001).

IL-6 PCR results: The one-way ANOVA showed a significant difference (F(4,25)=14; p<0.0001).

TNF-α WB results: The one-way ANOVA showed a significant difference (F(4,15)=11; p<0.001).

Incretin analogues protect dopaminergic neurons against enhanced 6-OHDA Induced α-Synuclein expression.

To further evaluate α-Synuclein (α-Syn) accumulation, we detected its expression in the substantia nigra through western blot assay. As shown in Figure 6, compared with the sham-surgery group, there was a significant increase of α-syn in the substantia nigra of 6-OHDA treated rats. However, DA5-CH treatment reduced α-Syn accumulation back to control levels. Exendin-4 showed a reduced effect compared to DA5-CH, but was more effective than Tirzepate (see Table 11).

The one-way ANOVA showed a significant difference (F(4,15)=47; p<0.001).