Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats

In pre-hypertension, moderate control of blood pressure (BP) can be obtained by a nutritional approach. The effects of a diet enriched with defatted larvae of the mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (TM) endowed with ACE inhibitory activity was studied in both spontaneously hypertensive rats (SHR) and in the age-matched normotensive Wistar Kyoto strain. These were fed for 4 weeks with standard laboratory rodent chow supplemented with or without TM or captopril. In SHR, the TM diet caused a significant reduction in BP, heart rate and coronary perfusion pressure, as well as an increase in red blood cell glutathione/glutathione disulphide ratio. Rat brain slices of SHR were more resistant to oxidative stress and contained lower levels of inflammatory cytokines, while vascular and liver enzyme-activities were not affected. These results suggest that TM can be considered a new functional food that can lower BP in vivo and thus control cardiovascular-associated risk factors such as hypertension.


Introduction 4. Line 44: ……Hypertension is the main risk factor for pathologies such as coronary artery disease……Pathology is the study of the way diseases and illnesses develop. Authors should avoid use this term as a synonym of disease.
We agree with the Referee and the text has been amended accordingly.
Methods 5. Line 158 : …..Heart contractility was measured as left ventricle pressure (LVP= left ventricular systolic pressure minus left ventricular diastolic pressure)…. I suggest that authors name it as …developed left ventricular systolic pressure, as it is not the absolute systolic pressure, but the systolic minus diastolic pressure.
We agree with the point raised and thus LVP has been replaced by LVDP in text, Figures and their legend.
Also, authors should inform the value of diastolic pressure in the left ventricle, as diastolic pressure is an important determinant of systolic pressure. This is described by Starling Law. That states the systolic pressure increases in response to an increase in the diastolic pressure or diastolic volume, before contraction (the end diastolic volume), when all other factors remain constant. It is important considering the ability of the heart to change its force of contraction in response to changes in diastolic pressure or volume. Please, inform the diastolic pressure or volume in the hearts.
According to referee's suggestion we have measured the diastolic pressure (see Figure below

LVEDP (mmHg)
Effects of the feeding for 4 weeks with standard laboratory rodent chow (SD) or SD supplemented with either Tenebrio molitor (TM) or captopril (C) on left ventricular end diastolic pressure (LVEDP) on isolated heart. Data are reported as mean±SEM. The perfusion solution was continuously gassed with 5% CO2 and 95% O2 at a constant temperature of 37°C to maintain an appropriate pH of 7.4. In S7 file is now reported that bubbling was continuous, being this not clearly stated. pH was checked several times, at the beginning, during and at the end of the experimental session, and it was found to be constant. EDTA Ca2+ buffering capacity was exploited to ensure a constant and controlled free Ca2+ concentration throughout the experimental session.
8. Line 116: why authors measured brain ACE activity and not cardiac? Also, why measure brain IL-6, IL1B and TNP and not vascular iNOS expression to correlate it with those pro-inflammatory mediators?
Cardiac ACE activity was not assessed, as heart were used to perform the experiments with isolated organ. As previously outlined, however, we are planning a novel in vivo and ex-vivo study, which will be focused on the changes induced at cardiovascular level by the proteins from defatted larvae of the mealworm in hypertensive rats, with particular regard to the role played by vascular NO, iNOS, eNOS and nNOS expression and inflammation (see also the answer to point 9 We thank the Referee for his suggestion. The vasoreactivity to phenylephrine has been analysed and discussed and now is included in the revised Supporting Figure 1, panel a. However, as TM supplementation did not improve either endothelium-dependent relaxation or smooth muscle sensitivity to phenylephrine, we believe that an in depth analysis of eNOS/NADPH oxidase expression as well as ROS production in aorta rings would not help clarifying the mechanism(s) underpinning the beneficial effect of TM treatment on systemic blood pressure and, therefore, has not been executed.
Results and Discussion 10. Isolated heart preparation and perfusion was described at methods section, but no results were presented at Results section. Please include it.
In In paragraph "Effects of TM on cardiovascular mechanics" was already stated "Regarding heart function, no type of diet fed to WKY or SHRs affected LVDP or heart rate as evaluated by the Langendorff perfused heart model. SHRs fed the standard diet showed higher coronary perfusion pressure than did the matched normotensive strain, with both captopril-and TM -supplemented diets bringing this parameter back to control values (Fig 2, panel d). We agree with the Referee that the original sentence might be ambiguous, and thus it was modified accordingly as reported below:

Lines
A feature of particular relevance shown by TM was its ability to prevent the marked increase in blood pressure and heart rate observed in SHRs, known to be associated with vascular and heart protection.
Reviewer #2: The manuscript "Antihypertensive, cardio-and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats" is very well written, and easy to understand. The topic is interesting and important because it deals with potential functional food as a source of alternative therapies for pre-hypertensive patients. Methods section provide enough details for the general reader to repeat the experiments. Аuthors report that diet enriched with defatted larvae of the mealworm in SHR caused a significant reduction of systolic blood pressure, heart rate and coronary perfusion pressure, also increased ACE inhibitory activity in plasma, and decreased inflammatory cytokines IL-1β and TNFα levels in brain homogenates. Further, hypertensive rat brain slices were more resistant to oxidative stress. I suggest that in the future studies focus is on the mechanistic investigations and explaining in more detail the cellular and molecular mechanisms of action of proteins from defatted larvae of the mealworm in hypertensive rats.
We thank the Referee for the suggestion that will be followed in the next study focused on the mechanisms responsible for the effects that the proteins from defatted larvae of the mealworm exert at cardiovascular level in hypertensive rats.