Effect of Lactobacillus rhamnosus (NR_113332.1) on Creatine Kinase and Lactate Dehydrogenase Levels in Orthrotopically Transplanted EDL Muscle of Mice

Male albino mice were purchased and housed 15 days before the experiment at 25±5 oC with a roughly light and dark cycle of 12:12h. They were provided free access to water and pelleted food to minimise the cage effect. After adopting the local environmental condition animals were randomly divided into three groups (Intact control, Control, Probiotic), each containing 6 animals. EDL muscle were orthrotransplant in right legs of the mice in groups 2 and 3 under ether anesthesia.

After surgeries, probiotic (Lactobacillus rhamnosus NR_113332.1) dose was administered orally via a gavage method on daily basis. Each animal in control group was given a dose of 200µL of saline on daily basis for 3, 5, 7 and 14 days. Whereas probiotic group was given a dose of 1x108 C.F.U. of the probiotic (NR_113332.1)/200ul/animal/day for 3, 5, 7 and 14 days after surgeries.

For preparation of probiotic dose ten autoclaved vials containing 9 mL of sterile MRS broth (Biolife, Italiana), and added 1 mL of cultural broth of Lactobacillus rhamnosus in vial label as one, and then serially diluted to vial labelled as ten. Then 1mL was taken from each serially diluted broth vials to measure its optical density and 0.1mL was taken from each vial and spread over MRS agar plates. Lactobacillus rhamnosus showed 1 × 108 C.F.U. mL−1 at 0.5 optical density when measured at 600nm using spectrophotometer (V-M5). Every time culture broth was diluted with un-inoculated broth until obtained 0.5 optical density. Diluted broth with optical density 0.5 was centrifuged to have C.F.U. 1× 108 mL−1 following the procedure by Hasan et al. (2022).

For collection of blood and tissue samples, animals were killed with overdose of ether. Grafted as well as non-grafted EDL muscles were recovered. Blood was collected from each mouse by direct puncturing of the heart.

Serum was seperated from the blood and used for the estimation of CK and LDH activities with the help of commercially available bio-kits (Merck, Pvt, Ltd) in automatic bioanalyzer (Erba Chem 5 V3, Germany).

The data were analyzed by two-way analysis of variance (ANOVA) using MINITAB-16 software. The mean was compared by using Tukey multiple range test. Differences between means ± SEM were considered significant at P < 0.05.

Following the grafting of EDL muscles there were no signs of sickness, diarrhea and aggressive behavior for both in the control as well as probiotic supplemented groups throughout the experimental period. All the experimental animals remained alive during the observational period. All animals were recovered after 3, 5, 7 and 14 days after surgeries, whereas intact control group was recovered at 0 day. At the time of recovery there was no apparent difference in size and morphology of visceral organs of probiotic treated and control mice were noticed.

Figure 1 shows the effect of probiotic on the level of CK and LDH in regenerating EDL muscle of mice. Significant increases in CK during the regenerative phases of control mice over the probiotic treated group were recorded. The control mice group had CK activity of 625.00 ± 184.68 and 354.67 ± 13.92 U/L on day 5 and 14 days of post grafting. Whereas the corresponding value in the probiotics treated group of mice were 558.67 ± 125.24 and 255.33 ± 12.88 (U/L), respectively (P<0.001). The intact control group showed 243.33 ± 19.10 U/L of CK activity peaked on day 5, which was 157.2% for control and 129.6% for probiotic treated group. Whereas, day 14 post-transplantation corresponding values for control and probiotics were 45.6% and 4.9%, respectively. Muscle grafting increased serum CK level despite from the fact that probiotic treatment tended to minimize the injury effect.

LDH level in blood increases with response to increase in muscle damage. Value of control EDL grafted mice group at 7 and 14 days of regeneration were 258.3 ± 42.1 and 208.7 ± 11.6 (mg/dl), respectively. Whereas probiotic group had the corresponding values of 230.00 ± 65.6 and 172.7 ± 26.00 (mg/dl) (P<0.016). Percentage increase was calculated from intact control which had value 169.67 ± 9.74 mg/dl. Control group represent highest increase in lactate level which was observed at day 7 of regeneration which was 52.27% but in probiotic supplemented group the peak 35.5% was observed at day 5 of post-transplantation. Whereas, at 14-days post-transplantation corresponding values for control and probiotics were 23.0% and 1.7 %, Results showed positive effect of probiotic supplementation reducing the serum lactate level as compared to control group (Fig. 1).

Probiotic treatment tended to minimize the injury effect in terms of creatine kinase at first as well as last sampling period. Presence of CK and LDH in blood stream of an organism is indicator of tissue damage (Foschini and Prestes, 2007). Creatine kinase is normally found in the muscles tissues but this enzyme can leak and be present in the bloodstream owing to the injury to the epimysium membrane surrounding the muscles. That’s why elevated CK levels is suggestive of muscular injury and is considered a marker of muscle damage. It is well documented that probiotics reduce levels of CK, LDH and exhibit a therapeutic impact compared with protective impact. Many authors have reported that different Lactobacillus species significantly reduce CK activity (Chen et al., 2016; Al-Orf et al., 2018; Al-Osaimi et al., 2018). Highest % increase in LDH level in control group was observed at day 7 day of regeneration which was 52.27% but in probiotic supplemented group the peak 35.5% was observed at day 5 of post-transplantation when compared with the value of intact control. The shifting of the peak of LDH level at an early phase and its regression in the later phase, is a good indicator of early occurring of degeneration and regeneration processes with EDL muscle grafts of the Lactobacillus rhamnosus (NR_113332.1) supplemented mice than the control grafted mice.

Delay in maximal elevations of CK and LDH contents may be caused by increasing membrane permeability due to secondary or delayed onset damage as a result of increasing Ca2+ leakage into the muscle, and thus increased calpain activity and further reductions in membrane integrity (Gissel and Clausen, 2001). Furthermore, the exercise-induced muscular activity release of predominantly cytoplasmic CK can be due to final death of the muscle fibre which result in temporary muscle fibre damage accompanied by membrane leakage (McNeil and Khakee, 1992), this process does not necessarily reflect myofibrillar disruption. Indeed, any correlation between leakage of muscle enzymes and force production is a complex process, as it is well established that the greatest elevation in plasma enzyme levels occurs in the days following injury at a time when force has considerably recovered (Chen and Hsieh, 2001; Beaton et al., 2002). However, Friden and Lieber (2001) suggested that increase in serum CK levels may provide a gross indication that skeletal muscle injury has occurred, differences between serum CK levels do not necessarily provide information regarding the extent of muscle damage.

This study showed that the probiotics supplementation after EDL muscle transplantation helped to reduce the level of CK and LDH than that of control group. The study also revealed that treatment with probiotics caused CK and LDH increases during early stages of regeneration with comparatively lower values than control group.

There study received no external funds.

Study was approved by the Punjab University Institutional Ethics Review Board vide letter No.D/68/FIMS.

The current study was approved by the Punjab University Ethics Review Board.

The authors have declared no conflict of interest.